Senecavirus a immunogenic compositions and methods thereof

ABSTRACT

The present invention relates to killed/inactivated and/or recombinant Senecavirus A immunogenic compositions and vaccines, and methods of preventing or treating animals in need of with such an immunogenic compositions and vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 16/629,525, filed on Jan. 8, 2020, which is a 371 of international PCT/US2018/041321, filed Jul. 9, 2018, which claims the benefit of priority to U.S. Provisional Patent Application No. 62/590,209 filed on Nov. 22, 2017, and which claims the benefit of priority to U.S. Provisional Patent Application No. 62/531,578 filed on Jul. 12, 2017, which are incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a sequence listing in accordance with 37 C.F.R. 1.821-1.825. The sequence listing accompanying this application is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION A. Field of the Invention

The present invention relates to Seneca Valley Virus A or Senecavirus A (SVA) and its use as an immunogenic composition or vaccine to treat animals affected by Senecavirus A.

B. Description of the Related Art

SVA is a non-enveloped, single-stranded, positive-sense RNA virus within the family Picornaviridae. Foot and Mouth Disease Virus (FMDV) and swine vesicular disease virus (SVDA) are also member of family Picornaviridae.

The virus was originally discovered as a contaminant from cell-culture medium (See, Hales, L. M., et al., Complete genome sequence analysis of Seneca Valley virus-001, a novel oncolytic picornavirus. J Gen Virol, 2008. 89(Pt 5): p. 1265-75, incorporated by reference in its entirety); however, neutralizing antibodies against the virus have been detected in swine, bovine, murine and humans (See, Knowles, N. J., et al., Epidemiology of Seneca Valley Virus: Identification and Characterization of Isolates from Pigs in the United States, in The Northern Lights EUROPIC 2006—14th meeting of the European Study Group on the Molecular Biology of Picornaviruses. 2006: Saairselka, Inari, Finland, incorporated by reference in its entirety). Reported clinical signs following infection include vesicular lesions on the snout and coronary band, acute lameness, ulceration of the coronary band and sloughing of the hoof (Singh, K., et al., Seneca Valley Virus and vesicular lesions in a pig with idiopathic vesicular disease. J Vet Sci Technol, 2012. 3(6) and Pasma, T., S. Davidson, and S. L. Shaw, Idiopathic vesicular disease in swine in Manitoba. CVJ, 2008. 49: p. 84-85, both incorporated by reference in their entirety). In 2016, Koch's postulate was fulfilled when a cell culture-propagated SVA isolate was used to inoculate conventional animals and vesicular lesions were observed four days post inoculation (Montiel, N., et al., Vesicular Disease in 9-Week-Old Pigs Experimentally Infected with Senecavirus A. Emerg Infect Dis, 2016. 22(7): p. 1246-8, incorporated by reference in its entirety).

U.S. Pat. No. 8,039,606 describes the use of a Seneca Valley Virus to treat tumors. However, this Seneca Valley virus differs from the SVA of the present invention.

There has been an unexplained increase in cases of SVA in the US, Canada, Australia, Italy, New Zealand and Brazil. Further, because of the similarity of clinical signs to FMDV, this virus is of interest to the swine industry. The Center for Veterinary Biologics (CVB) notice 16-03 confirmed that the CVB was interested in licensing biologics and/or prophylactics for SVA virus.

SUMMARY OF THE INVENTION

The present invention provides immunogenic compositions, vaccines, and related methods that overcome deficiencies in the art. The compositions and methods provide immunogenic compositions which include inactivated/killed and/or recombinant forms of a non-enveloped (+) single-stranded RNA virus of SVA. In particular, the application provides a vaccine for generating an immune response in porcine for protection against diseases associated with Senecavirus A. The present Senecavirus A isolate NAC #20150909 (SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3) was isolated from vesicular fluid collected from 5 month old swine exhibiting vesicular lesions on the snout and coronary band.

Immunogenic compositions and vaccines of the invention comprise SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3.

Exemplary compositions of the invention comprise the polypeptide sequences of SEQ ID NO: 3, or fragments thereof that are immunoreactive to SVA.

Immunogenic compositions and vaccines of the invention comprise a SVA antigen, expressed in one non-limiting example in insect cells via a recombinant baculovirus expressing a modified SVA P1, 2A, partial 2B and 3B, and 3C protease, e.g., modified SVA nucleic acid sequence (SEQ ID NO:18) encoding amino acid sequence (SEQ ID NO: 19) and typically also includes an adjuvant. The vaccine may also include other components, such as preservative(s), stabilizer(s) and antigens against other porcine pathogens.

A preferred P1-2A-P3 nucleic acid sequence suitable for use in the invention is a polynucleotide encoding a P1-2A-P3polypeptide, said polynucleotide having at least at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%. 99.9% sequence identity to SEQ ID NO: 18, 20, 22, 24, 32, and 33. “As used herein, it is in particular understood that the term “sequence identity to SEQ ID NO:X” or “identical SEQ ID NO:X”, respectively, is equivalent to the term “sequence identity with the sequence of SEQ ID NO:X over the length of SEQ ID NO: X” or “identical to the sequence of SEQ ID NO:X over the length of SEQ ID NO: X”, respectively, wherein in this context “X” is any integer selected from 18, 20, 22, 24, 32, and 33.”

A preferred P1-2A-P3 polypeptide suitable for use in the invention is the polypeptide having the sequence set out in SEQ ID NO: 19, 21, 23, 25, 27, and 29 having at least 80% homology with SEQ ID NO: 19, 21, 23, 25, 27, and 29 for example at least 85% homology with SEQ ID NO: 19, 21, 23, 25, 27, and 29, such as a least 85% homology with SEQ ID NO: 19, 21, 23, 25, 27, and 29, such as at least 90% homology with SEQ ID NO: 19, 21, 23, 25, 27, and 29 , for example at least 95%, at least 98% or at least 99% homology SEQ ID NO: 19, 21, 23, 25, 27, and 29.

In another aspect the invention provides nucleic acid sequences that encode one or more polypeptides, antibody constructs, or antibody conjugates. The gene sequences coding for the polypeptides comprise a nucleic acid sequence that is at least 95%, 90%, 85%, or even 80% homologous to and/or identical with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or fragments thereof coding for a polypeptide that is immunoreactive to SVA. Exemplary nucleic acid sequences of the invention include any one of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and fragments thereof that encode a polypeptide that is immunoreactive to SVA.

Moreover a polypeptide of the invention as used herein includes but is not limited to a polypeptide that comprises:

-   i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; -   ii) a polypeptide that is at least 80% homologous to and/or     identical with a polypeptide of i); -   iii) a fragment of the polypeptides of i) and/or ii); -   iv) a polypeptide of i) or ii); -   v) a fragment of iii) or iv) comprising at least 5, preferably 8,     more preferably 10, even more preferably 15 contiguous amino acids     included in the sequences of SEQ ID NO: 3; -   vi) a polypeptide that is encoded by a polynucleotide comprising the     sequence of SEQ ID NO: 1 or 2; -   vii) a polypeptide that is encoded by a polynucleotide that is at     least 80% homologous to or identical with polynucleotides of vi); -   viii) a protein fragment that is encoded by a polynucleotide that     comprises at least 15, preferably 24, more preferably 30, even more     preferably 45 contiguous nucleotides included in the sequences of     SEQ ID NO: 1 or SEQ ID NO: 2.

Immunogenic compositions of the invention which comprise at least one or more SVA polypeptides as defined herein may further comprise a physiologically-acceptable vehicle such as a pharmaceutically or veterinarily acceptable carrier, adjuvant, or combination thereof.

Any of the SVA polypeptides provided herewith or any immunogenic compositions comprising one or more of these SVA polypeptides provided herewith can be used as a medicament, preferably as a vaccine or immunogenic composition, most preferably for the prophylaxis or treatment of a subject against a SVA infection.

Those of skill in the art will understand that the compositions used herein may incorporate known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, e.g. saline or plasma protein solutions, are readily available. In addition, the immunogenic and vaccine compositions of the present invention can include veterinary-acceptable carriers, diluents, isotonic agents, stabilizers, or adjuvants.

Methods of the invention include, but are not limited to, a method of provoking an immune response against an SVA infection in a subject comprising the step of administering to the subject an immunogenic composition comprising one or more SVA polypeptides as defined herein. Compositions of the invention may be used to treat or alternatively to prevent an SVA infection. Preferably, such immune response reduces the incidence of or severity of one or more clinical signs associated with or caused by the infection with SVA serotypes.

Herein, suitable subjects and subjects in need to which compositions of the invention may be administered include animals in need of either prophylactic or treatment for a viral associated infection, disease, or condition. Animals in which the immune response is stimulated by use of compositions or methods of the invention include livestock, such as swine, bovines, goats, and sheep. Preferred animals include porcines, murids, equids, lagomorphs, and bovids. Most preferably, an immune response is stimulated in swine.

The invention also provides a method of reducing the incidence of or severity of one or more clinical signs associated with or caused by SVA infection, comprising the step of administering an immunogenic composition of the invention that comprises one or more SVA peptides as provided herewith and preferably a carrier molecule, such that the incidence of or the severity of a clinical sign of the SVA infection is reduced by at least 10%, preferably at least 20%, even more preferred at least 30%, even more preferred at least 50%, even more preferred at least 70%, most preferred at least 100% relative to a subject that has not received the immunogenic composition as provided herewith.

Such clinical signs include vesicular disease such as open or closed blisters or lesions located on the snout, oral mucosa, and/or at the junction where the skin and the hoof wall meet (Coronary band), nail bed hemorrhages, sudden lameness with redness and swelling at or around the coronary band; and breeding females that are suddenly off feed, lethargic, anorexic and/or have a fever up to 105° Fahrenheit (˜40.6° Celsius).

There appears to be a short term (4-10 days) increase in mortality in neonatal piglets (less than 7 days) that may or may not have diarrhea associated with it. Morbidity and mortality estimates are 30-70% for a short time period. It is usually upon investigation of the increase in neonatal mortality, that the vesicular lesions in the breeding age animals are noted. This type of infection in swine resulting in snout and coronary band vesicles has also been termed idiopathic vesicular disease in swine.

According to a further aspect, the present invention also relates to a method for the prophylaxis of an SVA infection, wherein said SVA infection may be caused by Seneca Valley Virus A, comprising the step of administering an immunogenic composition of the invention that comprises one or more SVA peptides as provided herewith.

The invention also provides a method of preparing any of the immunogenic compositions provided herewith that method comprises mixing one or more SVA peptides as provided herewith with a carrier molecule, preferably such that the one or more SVA peptides and carrier molecule are covalently coupled or conjugated to one another. Such conjugates may be multivalent or univalent. Multivalent compositions or vaccines include an immuno-conjugation of multiple SVA peptides with a carrier molecule. In a further aspect, the invention provides a method of producing one or more SVA peptides that method comprises transforming a host cell, preferably a prokaryotic cell such as E. coli with a nucleic acid molecule that codes for any of the SVA peptides as provided herewith. Alternatively, the host cell may be a eukaryotic cell such as an animal cell, protist cell, plant cell, or fungal cell. Preferably the eukaryotic cell is a mammalian cell such as CHO, BHK or COS, or a fungal cell such as Saccharomyces cerevisiae, or an insect cell such as Sf9.

Another aspect of the invention provides a method of producing one or more SVA peptides that induce an immune response against SVA. This comprises culturing a transformed expression vector coding for and expressing one or more SVA peptides disclosed herein. The expressed proteins are either retained by the expression organism or secreted into the culture medium. Expression is conducted under conditions sufficient to produce a SVA peptide capable of inducing an immune response to SVA.

A method of producing a recombinantly expressed P1-2A-P3 antigen vaccine generated in insect cells via a recombinant baculovirus expressing a modified SVA P1-2A-P3 protein is also provided. The method in one exemplary embodiment includes cloning the SVA P1-2A-P3 sequence into a vector pVL1393 (BD Biosciences) and co-transfect Sf9 insect cells. For the inactivated recombinant SVA material, SVA baculoviral harvest was inactivated for 24 hours using 5 mM BEI, clarified and 0.45 μm filtered. Typically, the inactivated virus is further processed, e.g., by concentration and blending with other components, to produce a commercial formulation. For example, the fluids containing the inactivated virus may be concentrated and blended with an adjuvant and/or antigen(s) to one or more other porcine pathogens.

Methods of making compositions of the invention may further comprise mixing the conjugate of one or more SVA peptides or inactivated whole-virus preparations and a carrier molecule with a physiologically-acceptable vehicle such as a pharmaceutically- or veterinary-acceptable carrier, adjuvant, or combination thereof. Those of skill in the art will recognize that the choice of vehicle, adjuvant, or combination will be determined by the delivery route, personal preference, and animal species among others.

In another aspect, the invention provides a method of diagnosing a SVA infection in a subject. That method comprises providing one or more SVA peptides; contacting the one or more SVA peptides with a sample obtained from the subject; and identifying the subject as having an SVA infection if an antibody capable of binding the one or more SVA peptides is detected in the sample.

In another respect, the invention provides a method of ascertaining that a subject has been previously exposed to a SVA infection and is able to express an immune response to SVA. That method comprises providing one or more SVA peptides; contacting the one or more SVA peptides with a sample obtained from the subject; and identifying the subject as having a SVA infection if an antibody capable of binding the one or more SVA peptides is detected in the sample.

The invention also provides kits that comprise an immunogenic composition that comprises one or more SVA peptides, preferably together with a carrier molecule; a container for packaging the immunogenic composition; a set of printed instructions; and a dispenser capable of administering the immunogenic composition to an animal. Optionally, the one or more SVA peptides and the carrier molecule may be packaged as a conjugate or as separate compounds. When supplied separately, a means of conjugating the one or more SVA peptides and carrier molecule, as well as appropriate printed instructions, is also supplied.

The invention also provides kits for vaccinating an animal comprising a set of printed instructions; a dispenser capable of administering the immunogenic composition provided herewith comprising one or more SVA peptides to an animal; and wherein at least one of SVA peptides effectively immunizes the animal against at least one disease associated with SVA infection. Preferably, the one or more SVA peptides are selected from those provided herewith. Kits of the invention may further comprise a veterinary acceptable carrier, adjuvant, or combination thereof.

The dispenser in a kit of the invention is capable of dispensing its contents as droplets; and the immunogenic composition comprises the SVA peptides as provided herewith included in the kit is capable of reducing the severity of at least one clinical sign of a SVA infection when administered intranasally, orally, intradermally, or intramuscularly to an animal. Preferably, the severity of a clinical sign is reduced by at least 10% preferably by at least 20%, even more preferred by at least 30%, even more preferred by at least 50%, even more preferred by at least 70%, most preferred by at least 100% as compared to an untreated, infected animal.

Methods for the treatment or prophylaxis of infections caused by SVA are also disclosed. The method comprises administering an effective amount of the immunogenic composition of the present invention to a subject, wherein said treatment or prophylaxis is selected from the group consisting of reducing signs of SVA infection, reducing the severity of or incidence of clinical signs of SVA infection, reducing the mortality of subjects from SVA infection, and combinations thereof.

Compositions of the invention further comprise a veterinarily acceptable carrier, adjuvant, or combination thereof. Such compositions may be used as a vaccine and comprise an inactivated vaccine. Such vaccines elicit a protective immunological response against at least one disease associated with SVA.

Those of skill in the art will understand that the compositions used herein may incorporate known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, e.g., saline or plasma protein solutions are readily available. In addition, the immunogenic and vaccine compositions of the present invention can include pharmaceutical- or veterinary-acceptable carriers, diluents, isotonic agents, stabilizers, or adjuvants.

Methods of the invention may also comprise admixing a composition of the invention with a veterinarily acceptable carrier, adjuvant, or combination thereof. Those of skill in the art will recognize that the choice of carrier, adjuvant, or combination will be determined by the delivery route, personal preference, and animal species among others.

Methods for the treatment or prophylaxis of infections caused by SVA are also disclosed. The method comprises administering an effective amount of the immunogenic composition of the present invention to an animal, wherein said treatment or prophylaxis is selected from the group consisting of reducing signs of SVA infection, reducing the severity of or incidence of clinical signs of SVA infection, reducing the mortality of animals from SVA infection, and combinations thereof.

Preferred routes of administration include intranasal, oral, intradermal, and intramuscular. Administration via the intramuscular route, most preferably in a single dose, is preferred. The skilled artisan will recognize that compositions of the invention may also be administered in two or more doses, as well as, by other routes of administration. For example, such other routes include subcutaneously, intracutaneously, intravenously, intravascularly, intraarterially, intraperitoneally, intrathecally, intratracheally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily, or intravaginally. Depending on the desired duration and effectiveness of the treatment, the compositions according to the invention may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.

The invention also provides kits for vaccinating an animal comprising a set of printed instructions; a dispenser capable of administering a vaccine to an animal; and at least one isolate from a SVA culture. Kits of the invention may further comprise a veterinarily acceptable carrier, adjuvant, or combination thereof.

The dispenser in a kit of the invention is capable of dispensing its contents as droplets; and the isolate included in the kit is capable of reducing the severity of at least one clinical sign of a SVA infection when administered intranasally, orally, intradermally, or intramuscularly to an animal. In some kits, the isolate is also capable of reducing the severity of at least one clinical sign of a SVA infection. Preferably, the severity of a clinical sign is reduced by at least 10% as compared to an untreated, infected animal.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the SVVP13C construct design (A) (SEQ ID NO:18) and SVVP 13C-CO (codon optimized) construct design (B) (SEQ ID NO:20) for the baculovirus expression system.

FIG. 2 shows the SVVP1-His-sIRES-SVV3C construct design (A) (SEQ ID NO:32) and SVVP1CO-His-sIRES-SVV3C (codon optimized) construct design (B) (SEQ ID NO:33) for the baculovirus expression system.

FIG. 3 shows SVVP13C VP3/VP1 construct design (A) (SEQ ID NO:22) to mutate the VP3/VP1 cleavage site and SVVP13CD construct design (B) (SEQ ID NO:24) for the baculovirus expression system

FIG. 4 shows Western blots of BaculoFBU/SVVP13C(A) (SEQ ID NO:18) and BaculoFBU/SVVP13C-CO (B) (SEQ ID NO:20) supernatant samples compared to native SVV antigen detected with anti-alpha-SVV VP1, anti-alpha-SVV VP2 and anti-alpha-SVV VP3 cross-absorbed rabbit polyclonal antibodies. Lane A is the protein standard, Line 1 is SVV Baculo Harvest Supernatant, Line 2 is SVV Antigen (SEQ ID NO:3) and Lane 3 is Baculo Harvest Supernatant Negative Control. Expected Band Sizes: Full length SVV P1=around 95 kDa (SEQ ID NO:19, amino acids 1-859), VP1=around 29 kDa (SEQ ID NO:19, amino acids 596-859), VP2—around 32 kDa (SEQ ID NO:19, amino acids 73-356), VP3=around 26 kDa (SEQ ID NO:19, amino acids 357-595).

FIG. 5 shows western blots of native SVV virus sucrose fractions (A) and BauloFBU/SVVP13C sucrose fractions (B) with alpha-SVV VP1, alpha-SVV VP2 and alpha-SVV PV3 rabbit polyclonal antibodies. Lane A is the protein standard, Lanes 1-10 is sucrose fractions 1-10, N-BaculoFBU/No Insert Negative Control, P is Positive Control Native inactivated SVV and S is starting sample for sucrose gradient. Expected Band sizes: Full length SVV P1=around 95 kDa (SEQ ID NO:19, amino acids 1-859), VP1=around 29 kDa (SEQ ID NO:19, amino acids 596-859), VP2—around 32 kDa (SEQ ID NO:19, amino acids 73-356), VP3=around 26 kDa (SEQ ID NO:19, amino acids 357-595).

FIG. 6 shows Western blots of BaculoFBU/SVVP13C (SEQ ID NO:18) and BaculoFBU/VVP13C VP3/VP1 (SEQ ID NO:22) supernatant harvests with α-SVV VP1 (top row) and α-SVV VP3 (bottom row) rabbit polyclonal antibodies. Lane A—Protein Standard, Lane 1—SVV Baculo Harvest Supernatant Lane 2—SVV Antigen and Lane 3—Baculo Harvest Supernatant Neg. Control.

FIG. 7 shows Western Blots of BaculoFBU/VVP13C VP3/VP1 (SEQ ID NO:22) sucrose fractions with an anti-SVV VP1, anti-SVV VP2 and anti-SVV VP3 rabbit polyclonal antibodies. Lane A is the Protein Standard, N is BaculoFBU/No insert native control, P is positive control native inactivated SVV (SEQ ID NO:3), S is SVVP13C VP3/VP1 (SEQ ID NO:22,) pellet re-suspended in TBS and Lane 1-9 is sucrose fractions 1-9. Full length SVV P1=around 95 kDa (SEQ ID NO:19, amino acids 1-859), VP1=around 29 kDa (SEQ ID NO:19, amino acids 596-859), VP2—around 32 kDa (SEQ ID NO:19, amino acids 73-356), VP332 around 26 kDa (SEQ ID NO:19, amino acids 357-595).

FIG. 8 shows Western Blots of BaculoFBU/VVP13CD (SEQ ID NO:24) Supernatant Harvest detected with an anti-SVV VP1, anti-SVV VP2 and anti-SVV VP3 rabbit polyclonal antibodies. Lane A is the Protein Standard, Lane 1 is SVV Baculo Harvest Supernatant, Lane 2 is SVV Antigen (SEQ ID NO:3) and Lane 3 is Baculo Harvest Supernatant Negative Control. Expected Band Sizes: Full length SVV P1=around 95 kDa (SEQ ID NO:19, amino acids 1-859), VP1=around 29 kDa (SEQ ID NO:19, amino acids 596-859), VP2—around 32 kDa (SEQ ID NO:19, amino acids 73-356), VP3=around 26 kDa (SEQ ID NO:19, amino acids 357-595).

FIG. 9 shows Western Blots of BaluloFBU/SVVP13CD (SEQ ID NO:24) sucrose fractions with anti-SVV VP1, anti-SVV VP2, and anti-SVV VP3 rabbit polyclonal antibodies. Lane A is the Protein Standard, Lane 1 is SVV Baculo Harvest Supernatant, Lane N is BaculoFBU/No Insert Negative Control, P is Positive Control Native inactivated SVV (SEQ ID NO.3), S is SVVP13CD (SEQ ID NO.25) pellet re-suspended in TBA and Lane 1-10 is Sucrose Fractions 1-10. Expected Band Sizes: Full length SVV P1=around 95 kDa (SEQ ID NO:19, amino acids 1-859), VP1=around 29 kDa (SEQ ID NO:19, amino acids 596-859), VP2—around 32 kDa (SEQ ID NO:19, amino acids 73-356), VP3=around 26 kDa (SEQ ID NO:19, amino acids 357-595).

FIG. 10 shows Western Blots of BaculoFBU/SVVP13C (A) (SEQ ID NO:18), BaculoFBU/SVVP13CD (A) (SEQ ID NO:24), and BaculoFBU/SVVP13CVP3/VP1 (B) (SEQ ID NO:22). Day 3 soluble fractions with α-SVV VP1, α-SVV VP2 and α-SVV VP3 rabbit polyclonal antibodies. SDS-PAGE gel for BaculoFBU/SVVP13C and BaculoFBU/SVVP13CD (C). Lane A—Protein Standard, Lane P—Positive Control Native SVV Antigen, Lane 1—Day 3 Soluble Fraction SVVP13C, Lane 2—Day 3 Soluble Fraction SVVP13CD, Lane 3—Day 3 Soluble Fraction SVVP13C VP3/VP1 and Lane N—Negative Control BaculoFBU/No Insert Soluble Fraction. Expected Band Sizes: Full length SVV P1=around 95 kDa (SEQ ID NO:19, amino acids 1-859), VP1=around 29 kDa (SEQ ID NO:19, amino acids 596-859), VP2—around 32 kDa (SEQ ID NO:19, amino acids 73-356), VP3=around 26 kDa (SEQ ID NO:19, amino acids 357-595).

FIG. 11 shows Western blots of sucrose fractions of Day 3 Soluble BaculoFBU/SVVP13C (SEQ ID NO:18), BaculoFBU/SVVP13CD (SEQ ID NO:24) and BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) with anti-alpha-SVV VP1, anti-alpha-SVV VP2 and anti-alpha-SVV VP3 rabbit polyclonal antibodies. Lane A is Protein Standard, P is Positive Control Native Inactivated SVV, Lane 1-11 is Sucrose fractions 1-11 and N is Negative Control BaculoFBU/No Insert Soluble Fraction. Expected Band Sizes: Full length SVV P1=around 95 kDa (SEQ ID NO:19, amino acids 1-859), VP1=around 29 kDa (SEQ ID NO:19, amino acids 596-859), VP2—around 32 kDa (SEQ ID NO:19, amino acids 73-356), VP3=around 26 kDa (SEQ ID NO:19, amino acids 357-595)

FIG. 12 shows the least square mean rectal temperatures (°C) by study day and group.

FIG. 13 shows the group median log₁₀ genomic copies/mL SVA RNA in serum by group and day.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, protein chemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second Edition (1989); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Protein purification methods—a practical approach (E. L. V. Harris and S. Angal, eds., IRL Press at Oxford University Press); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular DNA, polypeptide sequences or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antigen” includes a mixture of two or more antigens; reference to “an excipient” includes mixtures of two or more excipients, and the like.

A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs at the time of filing. The meaning and scope of terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms such as “includes” and “included” is not limiting. All patents and publications referred to herein are incorporated by reference herein.

“Protection against disease”, “protective immunity”, “functional immunity” and similar phrases, means a response against a disease or condition generated by administration of one or more therapeutic compositions of the invention, or a combination thereof, that results in fewer deleterious effects than would be expected in a non-immunized subject that has been exposed to disease or infection. That is, the severity of the deleterious effects of the infection is lessened in a vaccinated subject. Infection may be reduced, slowed, or possibly fully prevented, in a vaccinated subject. Herein, where complete prevention of infection is meant, it is specifically stated. If complete prevention is not stated then the term includes partial prevention.

Herein, “reduction of the incidence and/or severity of clinical signs” or “reduction of clinical symptoms” means, but is not limited to, reducing the number of infected subjects in a group, reducing or eliminating the number of subjects exhibiting clinical signs of infection, or reducing the severity of any clinical signs that are present in one or more subjects, in comparison to wild-type infection. For example, it should refer to any reduction of pathogen load, pathogen shedding, reduction in pathogen transmission, or reduction of any clinical sign symptomatic of malaria Preferably these clinical signs are reduced in one or more subjects receiving the therapeutic composition of the present invention by at least 10% in comparison to subjects not receiving the composition and that become infected. More preferably clinical signs are reduced in subjects receiving a composition of the present invention by at least 20%, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%.

The term “increased protection” herein means, but is not limited to, a statistically significant reduction of one or more clinical symptoms which are associated with infection by an infectious agent, preferably SVA, respectively, in a vaccinated group of subjects vs. a non-vaccinated control group of subjects. The term “statistically significant reduction of clinical symptoms” means, but is not limited to, the frequency in the incidence of at least one clinical symptom in the vaccinated group of subjects is at least 10%, preferably 20%, more preferably 30%, even more preferably 50%, and even more preferably 70% lower than in the non-vaccinated control group after the challenge the infectious agent.

“Long-lasting protection” shall refer to “improved efficacy” that persists for at least 3 weeks, but more preferably at least 3 months, still more preferably at least 6 months. In the case of livestock, it is most preferred that the long lasting protection shall persist until the average age at which animals are marketed for meat.

An “immunogenic or immunological composition” refers to a composition of matter that comprises at least one SVA immunogenic composition, or immunogenic portion thereof, that elicits an immunological response in the host of a cellular or antibody-mediated immune response to the composition. In a preferred embodiment of the present invention, an immunogenic composition induces an immune response and, more preferably, confers protective immunity against one or more of the clinical signs of a SVA infection.

An “immunogenic” or “antigen” as used herein refer to a polypeptide or protein that elicits an immunological response as described herein. An “immunogenic” SVA protein or polypeptide includes the full-length sequence of any of the SVA identified herein or analogs or immunogenic fragments thereof. The term “immunogenic fragment” or “immunogenic portion” refers to a fragment or truncated and/or substituted form of an SVA that includes one or more epitopes and thus elicits the immunological response described herein. In general, such truncated and/or substituted forms, or fragments will comprise at least six contiguous amino acids from the full-length SVA protein. More preferably, the truncated or substituted forms, or fragments will have at least 10, more preferably at least 15, and still more preferably at least 19 contiguous amino acids from the full-length SVA protein. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known and described in the art, see e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and two-dimensional nuclear magnetic resonance. See Epitope Mapping Protocols, supra. Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol. and Cell Biol. 75:402-408; and Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-Jul. 3, 1998. (The teachings and content of which are all incorporated by reference herein.)

An “immune response” or “immunological response” means, but is not limited to, the development of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an immune or immunological response includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or a protective immunological (memory) response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in number of symptoms, severity of symptoms, or the lack of one or more of the symptoms associated with the infection of the pathogen, a delay in the of onset of viremia, reduced viral persistence, a reduction in the overall viral load and/or a reduction of viral excretion.

Herein, “specifically immunoreactive” refers to an immunoreactive protein or polypeptide that recognizes an antigen characteristic of SVA infection but does not react with an antigen characteristic of a strict challenge control.

As used herein, “a pharmaceutical- or veterinary-acceptable carrier” includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In some preferred embodiments, and especially those that include lyophilized immunogenic compositions, stabilizing agents for use in the present invention include stabilizers for lyophilization or freeze-drying.

In some embodiments, the immunogenic composition of the present invention contains an adjuvant. “Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.), JohnWiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). Exemplary adjuvants are the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name CARBOPOL® (Lubrizol)) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 971P. Most preferred is the use of Carbopol 971P. Among the copolymers of maleic anhydride and alkenyl derivative, are the copolymers EMA (Monsanto), which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof or stimulants of endogenous cytokine release, among many others.

It is expected that an adjuvant can be added in an amount of about 100 μg to about 10 mg per dose, preferably in an amount of about 100 μg to about 10 mg per dose, more preferably in an amount of about 500 μg to about 5 mg per dose, even more preferably in an amount of about 750 μg to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of about 0.01 to 50%, preferably at a concentration of about 2% to 30%, more preferably at a concentration of about 5% to 25%, still more preferably at a concentration of about 7% to 22%, and most preferably at a concentration of 10% to 20% by volume of the final product.

“Diluents” can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others.

“Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.

“Safety” refers to the absence of adverse consequences in a vaccinated animal following vaccination, including but not limited to: potential reversion of a bacterium-based vaccine to virulence, clinically significant side effects such as persistent, systemic illness or unacceptable inflammation at the site of vaccine administration.

The terms “vaccination” or “vaccinating” or variants thereof, as used herein means, but is not limited to, a process which includes the administration of an immunogenic composition of the invention that, when administered to an animal, elicits, or is able to elicit—directly or indirectly—an immune response in the animal against SVA.

“Mortality”, in the context of the present invention, refers to death caused by SVA infection, and includes the situation where the infection is so severe that an animal is euthanized to prevent suffering and provide a humane ending to its life.

Herein, “effective dose” means, but is not limited to, an amount of antigen that elicits, or is able to elicit, an immune response that yields a reduction of clinical symptoms in an animal to which the antigen is administered.

As used herein, the term “effective amount” means, in the context of a composition, an amount of an immunogenic composition capable of inducing an immune response that reduces the incidence of or lessens the severity of infection or incident of disease in an animal. Particularly, an effective amount refers to colony forming units (CFU) per dose. Alternatively, in the context of a therapy, the term “effective amount” refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity or duration of a disease or disorder, or one or more symptoms thereof, prevent the advancement of a disease or disorder, cause the regression of a disease or disorder, prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disease or disorder, or enhance or improve the prophylaxis or treatment of another therapy or therapeutic agent.

The term “fragment” refers to a fragment or truncated and/or substituted form of a SVA peptide or a gene coding for such SVA peptide that includes one or more epitopes and thus elicits the immunological response against SVA. Preferably, such fragment is a fragment or truncated and/or substituted form of any of the SVA peptides or any of the SVA genes provided herewith. In general, such truncated and/or substituted forms, or fragments will comprise at least six contiguous amino acids from the full-length SVA sequence. More preferably, the truncated or substituted forms, or fragments will have at least 10, more preferably at least 15, and still more preferably at least 19 contiguous amino acids from the full-length SVA sequence. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known and described in the art, see e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and two-dimensional nuclear magnetic resonance. See Epitope Mapping Protocols, supra. Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996), J. Immunol. 157:3242-3249; Suhrbier, A. (1997), Immunol. and Cell Biol. 75:402-408; and Gardner et al., (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-Jul. 3, 1998. (The teachings and content of which are all incorporated by reference herein.)

The term “variant” with respect to sequences (e.g., a polypeptide or nucleic acid sequence) is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein for the purposes of codon optimization. Generally, nucleotide sequence variants of the invention will have at least at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1% , 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%. 99.9% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters.

The term “immunoreactive to SVA” as used herein means that the peptide or fragment elicits the immunological response against SVA.

“Sequence homology”, as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homolog sequence comprises at least a stretch of 50, even more preferred of 100, even more preferred of 250, even more preferred of 500 nucleotides.

A “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

“Sequence Identity” as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% “sequence identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.

The terms “sequence identity” or “percent identity” are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions (i.e. overlapping positions)×100). Preferably, the two sequences are the same length.

A sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragment of the two sequences. Typically and preferred in the scope of the present invention, the comparison will be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

As used herein, it is in particular understood that the term “having at least X% sequence identity with the nucleic acid/amino acid sequence according to SEQ ID NO:Y” (or, alternatively, the term “having at least X% sequence identity with the nucleic acid/amino acid sequence of/set forth in SEQ ID NO:Y”) is equivalent to the term “having at least X% sequence identity with the nucleic acid/amino acid sequence according to SEQ ID NO:Y over the length of SEQ ID NO:Y” or to the term “having at least X% sequence identity with the nucleic acid/amino acid sequence according to SEQ ID NO:Y over the whole length of SEQ ID NO:Y”, respectively.

Vectors and methods for making and/or using vectors (or recombinants) for expression can be by or analogous to the methods disclosed in: U.S. Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, PCT publications WO 94/16716, WO 96/39491, WO 95/30018; Paoletti, “Applications of pox virus vectors to vaccination: An update, “PNAS USA 93: 11349-11353, October 1996; Moss, “Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety,” PNAS USA 93: 11341-11348, October 1996; Smith et al., U.S. Pat. No. 4,745,051 (recombinant baculovirus); Richardson, C. D. (Editor), Methods in Molecular Biology 39, “Baculovirus Expression Protocols” (1995 Humana Press Inc.); Smith et al., “Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector”, Molecular and Cellular Biology, December, 1983, Vol. 3, No. 12, p. 2156-2165; Pennock et al., “Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector, “Molecular and Cellular Biology March 1984, Vol. 4, No. 3, p. 406; EPA0 370 573; U.S. application Ser. No. 920,197, filed Oct. 16, 1986; EP Patent publication No. 265785; U.S. Pat. No. 4,769,331 (recombinant herpesvirus); Roizman, “The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors,” PNAS USA 93:11307-11312, October 1996; Andreansky et al., “The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors,” PNAS USA 93: 11313-11318, October 1996; Robertson et al., “Epstein-Barr virus vectors for gene delivery to B lymphocytes”, PNAS USA 93: 11334-11340, October 1996; Frolov et al., “Alphavirus-based expression vectors: Strategies and applications,” PNAS USA 93: 11371-11377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991; U.S. Pat. Nos. 5,591,439, 5,552,143; WO 98/00166; allowed U.S. application Ser. No. 08/675,556, and Ser. No. 08/675,566 both filed Jul. 3, 1996 (recombinant adenovirus); Grunhaus et al., 1992, “Adenovirus as cloning vectors,” Seminars in Virology (Vol. 3) p. 237-52, 1993; Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen Virol. 70, 42434; PCT WO 91/11525; Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993; and McClements et al., “Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease”, PNAS USA 93: 11414-11420, October 1996; and U.S. Pat. Nos. 5,591,639, 5,589,466, and 5,580,859, as well as WO 90/11092, WO 93/19183, WO 94/21797, WO 95/11307, WO 95/20660; Tang et al., Nature, and Furth et al., Analytical Biochemistry, relating to DNA expression vectors, inter alia. See also WO 98/33510; Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Pat. No. 4,945,050; Fischbachet al. (Intracel); WO 90/01543; Robinson et al., Seminars in Immunology vol. 9, pp. 271-283 (1997), (DNA vector systems); Szoka et al., U.S. Pat. No. 4,394,448 (method of inserting DNA into living cells); McCormick et al., U.S. Pat. No. 5,677,178 (use of cytopathic viruses); and U.S. Pat. No. 5,928,913 (vectors for gene delivery); as well as other documents cited herein.

Preferred viral vectors include baculovirus such as BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.), in particular provided that the production cells are insect cells. Although the baculovirus expression system is preferred, it is understood by those of skill in the art that other expression systems will work for purposes of the present invention.

B. Carriers Molecules

The carrier molecules to which the SVA peptides of the invention can be conjugated or covalently linked are preferably those described above. Preferred carriers for animal use are bovine serum albumin and Keyhole Limpet Hemocyanin. Protein carriers suitable for human use include tetanus toxoid, diphtheria toxoid, acellular pertussis vaccine (LPF toxoid), cross-reacting materials (CRM's) which are antigenically similar to bacterial toxins but are non-toxic by means of mutation. For example, CRM 197 obtained according to Pappenheimer, et al, Immunochemistry, 9, 891-906 (1972), and other bacterial protein carriers, for example meningococcal outer membrane protein may be used. Preferably, the carrier protein itself is an immunogen.

The SVA peptides of the invention may be covalently coupled to the carrier by any convenient method known to the art. While use of a symmetric linker such as adipic acid dihydrazide, as described by Schneerson et al, J. Experimental Medicine, 152, 361-376 (1980), or a heterobifunctional linker such as N-succinimidyl 3-(2-pyridyldithio) propionate as described by Fattom et al, Infection and Immunity, 56, 2292-2298 (1988) are within the scope of the invention, it is preferred to avoid the use of any linker but instead couple a SVA peptide of the invention directly to the carrier molecule. Such coupling may be achieved by means of reductive amination as described by Landi et al J. Immunology, 127, 1011-1019 (1981).

The size of the immunogenic composition, as defined by average molecular weight, is variable and dependent upon the chosen SVA peptide(s) and the method of coupling of the SVA peptide(s) to the carrier. Therefore, it can be as small as 1,000 daltons (10³) or greater than 10⁶ daltons. With the reductive amination coupling method, the molecular weight of the SVA peptide(s) is usually within the range of 5,000 to 500,000, for example 300,000 to 500,000, or for example 5,000 to 50,000 daltons.

Carrier molecules, i.e. peptides, derivatives and analogs thereof, and peptide mimetics that specifically bind a SVA peptide of the invention can be produced by various methods known in the art, including, but not limited to solid-phase synthesis or by solution (Nakanishi et al., 1993, Gene 137:51-56; Merrifield, 1963, J. Am. Chem. Soc. 15:2149-2154; Neurath, H. et al., Eds., The Proteins, Vol II, 3d Ed., p. 105-237, Academic Press, New York, N.Y. (1976), incorporated herein in their entirety by reference).

The SVA peptides of the invention or the antibodies or binding portions thereof of the present invention may be administered in injectable dosages by solution or suspension of in a diluent with a pharmaceutical or veterinary carrier.

Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population).

The vaccines of the invention may be multivalent or univalent. Multivalent vaccines are made from immuno-conjugation of multiple SVA peptides with a carrier molecule.

In one aspect, the SVA peptide compositions comprise an effective immunizing amount of the immunogenic conjugate, preferably in combination with an immunostimulant; and a physiologically acceptable vehicle. As used in the present context, “immunostimulant” is intended to encompass any compound or composition which has the ability to enhance the activity of the immune system, whether it is a specific potentiating effect in combination with a specific antigen, or simply an independent effect upon the activity of one or more elements of the immune response. Immunostimulant compounds include but are not limited to mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin, PLURONIC® polyols; polyanions; peptides; oil emulsions; alum, and MDP. Methods of utilizing these materials are known in the art, and it is well within the ability of the skilled artisan to determine an optimum amount of stimulant for a given vaccine. More than one immunostimulant may be used in a given formulation. The immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration preferably for administration to a mammal, especially a pig. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

C. Adjuvants

In order to further increase the immunogenicity of the immunogenic compositions provided herewith, and which contain one or more SVA peptides may also comprise one or more adjuvants.

The adjuvant may be purified by any of the techniques described previously or known in the art. The preferred purification technique is silica gel chromatography, in particular the “flash” (rapid) chromatographic technique, as described by W. Clark Still et al, J. Organic Chemistry, 43, 2923-2925 (1978). However, other chromatographic methods, including HPLC, may be used for purification of the adjuvant. Crystallization may also be used to purify the adjuvant. In some cases, no purification is required as a product of analytical purity is obtained directly from the synthesis.

The vaccine compositions of the invention are prepared by physically mixing the adjuvant with the SVA peptide(s) under appropriate sterile conditions in accordance with known techniques to produce the adjuvanted composition. Complexation of the SVA peptide(s) and the adjuvant is facilitated by the existence of a net negative charge on the conjugate which is electrostatically attracted to the positive charge present on the long chain alkyl compound adjuvant.

It is expected that an adjuvant can be added in an amount of about 100 μg to about 10 mg per dose, preferably in an amount of about 100 μg to about 10 mg per dose, more preferably in an amount of about 500 μg to about 5 mg per dose, even more preferably in an amount of about 750 μg to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of about 0.01% to 75%, preferably at a concentration of about 2% to 30%, more preferably at a concentration of about 5% to 25%, still more preferably at a concentration of about 7% to 22%, and most preferably at a concentration of 10% to 20% by volume of the final product.

D. Physiologically-Acceptable Vehicles

The vaccine compositions of this invention may be formulated using techniques similar to those used for other pharmaceutical polypeptide compositions. Thus, the adjuvant and SVA peptide(s), preferably conjugated to carrier molecule and/or admixed with an adjuvant may be stored in lyophilized form and reconstituted in a physiologically acceptable vehicle to form a suspension prior to administration. Alternatively, the adjuvant and conjugate may be stored in the vehicle. Preferred vehicles are sterile solutions, in particular, sterile buffer solutions, such as phosphate buffered saline. Any method of combining the adjuvant and the conjugate in the vehicle such that improved immunological effectiveness of the immunogenic composition is appropriate.

The volume of a single dose of the vaccine of this invention may vary but will be generally within the ranges commonly employed in conventional vaccines. The volume of a single dose is preferably between about 0.1 ml and about 3 ml, preferably between about 0.2 ml and about 1.5 ml, more preferably between about 0.2 ml and about 0.5 ml at the concentrations of conjugate and adjuvant noted above.

The vaccine compositions of the invention may be administered by any convenient means.

E. Formulations

Immunogenic conjugates comprising a SVA peptide coupled to a carrier molecule can be used as vaccines for immunization against SVA. The vaccines, comprising the immunogenic conjugate in a physiologically acceptable vehicle, are useful in a method of immunizing animals, preferably swine, for treatment or prevention of infections by SVA.

Antibodies generated against immunogenic conjugates of the present invention by immunization with an immunogenic conjugate can be used in passive immunotherapy and generation of anti-idiotypic antibodies for treating or preventing infections of SVA.

The subject to which the composition is administered is preferably an animal, including but not limited to cows, horses, sheep, pigs, poultry (e.g. chickens), goats, cats, dogs, hamsters, mice and rats, most preferably the mammal is swine.

The formulations of the invention comprise an effective immunizing amount of one or more immunogenic compositions or antibodies thereto and a physiologically acceptable vehicle. Vaccines comprise an effective immunizing amount of one or more immunogenic compositions and a physiologically acceptable vehicle. The formulation should suit the mode of administration.

The immunogenic composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The immunogenic composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

F. Effective Dose

The compounds described herein can be administered to a subject at therapeutically effective doses to treat SVA-associated diseases. The dosage will depend upon the host receiving the vaccine as well as factors such as the size, weight, and age of the host.

The precise amount of immunogenic conjugate or antibody of the invention to be employed in a formulation will depend on the route of administration and the nature of the subject (e.g., species, age, size, stage/level of disease), and should be decided according to the judgment of the practitioner and each subjects circumstances according to standard clinical techniques. An effective immunizing amount is that amount sufficient to treat or prevent a SVA infectious disease in a subject. An example of an appropriate dose is about 6 to 7 log TCID50/mL. Alternatively, effective doses may also be extrapolated from dose-response curves derived from animal model test systems and can vary from 0.001 mg/kg to 100 mg/kg.

Toxicity and therapeutic efficacy of compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in animals, especially swine. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in subjects. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Immunogenicity of a composition can be determined by monitoring the immune response of test subjects following immunization with the composition by use of any immunoassay known in the art. Generation of a humoral (antibody) response and/or cell-mediated immunity, may be taken as an indication of an immune response. Test subjects may include animals such as pigs, mice, hamsters, dogs, cats, rabbits, cows, horses, sheep, poultry (e.g. chickens, ducks, geese, and turkeys).

The immune response of the test subjects can be analyzed by various approaches such as: the reactivity of the resultant immune serum to the immunogenic conjugate, as assayed by known techniques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots, immunoprecipitation, etc.; or, by protection of immunized hosts from infection by the pathogen and/or attenuation of symptoms due to infection by the pathogen in immunized hosts as determined by any method known in the art, for assaying the levels of an infectious disease agent, e.g., the bacterial levels (for example, by culturing of a sample from the subject), or other technique known in the art. The levels of the infectious disease agent may also be determined by measuring the levels of the antigen against which the immunoglobulin was directed. A decrease in the levels of the infectious disease agent or an amelioration of the symptoms of the infectious disease indicates that the composition is effective.

The therapeutics of the invention can be tested in vitro for the desired therapeutic or prophylactic activity, prior to in vivo use in animals or humans. For example, in vitro assays that can be used to determine whether administration of a specific therapeutic is indicated include in vitro cell culture assays in which appropriate cells from a cell line or cells cultured from a subject having a particular disease or disorder are exposed to or otherwise administered a therapeutic, and the effect of the therapeutic on the cells is observed.

Alternatively, the therapeutic may be assayed by contacting the therapeutic to cells (either cultured from a subject or from a cultured cell line) that are susceptible to infection by the infectious disease agent but that are not infected with the infectious disease agent, exposing the cells to the infectious disease agent, and then determining whether the infection rate of cells contacted with the therapeutic was lower than the infection rate of cells not contacted with the therapeutic. Infection of cells with an infectious disease agent may be assayed by any method known in the art.

In addition, the therapeutic can be assessed by measuring the level of the molecule against which the antibody is directed in the animal model or human subject at suitable time intervals before, during, or after therapy. Any change or absence of change in the amount of the molecule can be identified and correlated with the effect of the treatment on the subject. The level of the molecule can be determined by any method known in the art.

After vaccination of an animal to a SVA infection using the methods and compositions of the present invention, any binding assay known in the art can be used to assess the binding between the resulting antibody and the particular molecule. These assays may also be performed to select antibodies that exhibit a higher affinity or specificity for the particular antigen.

G. Detection and Diagnostic Methods

Antibodies, or binding portions thereof, resulting from the use of SVA peptides of the present invention are useful for detecting in a sample the presence of SVA virus. This detection method comprises the steps of providing an isolated antibody or binding portion thereof raised against an SVA peptide of the invention, adding to the isolated antibody or binding portion thereof a sample suspected of containing a quantity of SVA, and detecting the presence of a complex comprising the isolated antibody or binding portion thereof bound to SVA.

The antibodies or binding portions thereof of the present invention are also useful for detecting in a sample the presence of a SVA peptide. This detection method comprises the steps of providing an isolated antibody or binding portion thereof raised against a SVA peptide, adding to the isolated antibody or binding portion thereof a sample suspected of containing a quantity of the SVA peptide, and detecting the presence of a complex comprising the isolated antibody or binding portion thereof bound to the SVA peptide.

Immunoglobulins, particularly antibodies, (and functionally active fragments thereof) that bind a specific molecule that is a member of a binding pair may be used as diagnostics and prognostics, as described herein. In various embodiments, the present invention provides the measurement of a member of the binding pair, and the uses of such measurements in clinical applications. The immunoglobulins in the present invention may be used, for example, in the detection of an antigen in a biological sample whereby subjects may be tested for aberrant levels of the molecule to which the immunoglobulin binds, and/or for the presence of abnormal forms of such molecules. By “aberrant levels” is meant increased or decreased relative to that present, or a standard level representing that present, in an analogous sample from a portion of the body or from a subject not having the disease. The antibodies of this invention may also be included as a reagent in a kit for use in a diagnostic or prognostic technique.

In one aspect, an antibody of the invention that immunospecifically binds to a SVA peptide may be used to diagnose, prognosis or screen for a SVA infection.

In another aspect, the invention provides a method of diagnosing or screening for the presence of a SVA infection or immunity thereto, comprising measuring in a subject the level of immunospecific binding of an antibody to a sample derived from the subject, in which the antibody immunospecifically binds a SVA peptide in which an increase in the level of said immunospecific binding, relative to the level of said immunospecific binding in an analogous sample from a subject not having the infectious disease agent, indicates the presence of SVA.

Examples of suitable assays to detect the presence of SVA peptides or antagonists thereof include but are not limited to ELISA, radioimmunoassay, gel-diffusion precipitation reaction assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay, protein A immunoassay, or immunoelectrophoresis assay.

Immunoassays for the particular molecule will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cultured cells, in the presence of a detectably labeled antibody and detecting the bound antibody by any of a number of techniques well-known in the art.

The binding activity of a given antibody may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

An additional aspect of the present invention relates to diagnostic kits for the detection or measurement of SVA. Kits for diagnostic use are provided, that comprise in one or more containers an anti-SVA peptide antibody, and, optionally, a labeled binding partner to the antibody. Alternatively, the anti-SVA peptide antibody can be labeled (with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety). Accordingly, the present invention provides a diagnostic kit comprising, an anti-SVA peptide antibody and a control immunoglobulin. In a specific embodiment, one of the foregoing compounds of the container can be detectably labeled. A kit can optionally further comprise, in a container, a predetermined amount of a SVA peptide recognized by the antibody of the kit, for use as a standard or control.

Preferred routes of administration include but are not limited to intranasal, oral, intradermal, and intramuscular. Administration via the intramuscular route, most preferably in a single dose, is desirable. The skilled artisan will recognize that compositions of the invention may also be administered in one, two or more doses, as well as, by other routes of administration. For example, such other routes include subcutaneously, intracutaneously, intravenously, intravascularly, intraarterially, intraperitoneally, intrathecally, intratracheally, intracutaneously, intracardially, intralobally, intramedullary, intrapulmonarily, and intravaginally. Depending on the desired duration and effectiveness of the treatment, the compositions according to the invention may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

SEQUENCE LISTING

This application contains a sequence listing. The sequence listing comprises the following sequences:

SEQ ID NO:1 denotes the RNA sequence of the near-complete genome of the SVA strain of the present invention.

SEQ ID NO:2 denotes a subset of the genomic RNA sequence that encodes the viral polyprotein (SVV antigen; SEQ ID NO:3).

SEQ ID NO:3 denotes the amino acid sequence of the viral polyprotein (SVV antigen) encoded by SEQ ID NO:2.

SEQ ID NO:4 denotes GenBank_KX377924.

SEQ ID NO:5 denotes GenBank_KT321458.

SEQ ID NO:6 denotes GenBank_KR063107.

SEQ ID NO:7 denotes GenBank_KR063108.

SEQ ID NO:8 denotes GenBank_KR063109.

SEQ ID NO:9 denotes GenBank_HJ999048.

SEQ ID NO:10 denotes GenBank_KC667560.

SEQ ID NO:11 denotes GenBank_GY488390.

SEQ ID NO:12 denotes GenBank_GV614995.

SEQ ID NO:13 denotes GenBank_DM060849.

SEQ ID NO:14 denotes GenBank_NC_011349.

SEQ ID NO:15 denotes GenBank_GY488390_CDS.

SEQ ID NO:16 denotes GenBank_GV614995_CDS.

SEQ ID NO:17 denotes GenBank_DM060849_CDS.

SEQ ID NO:18 denotes the nucleotide sequence of SVVP13C insert in construct BaculoFBU/SVVP13C (A).

SEQ ID NO:19 denotes the amino acid sequence of SVVP13C polyprotein expressed from BaculoFBU/SVVP13C.

SEQ ID NO:20 denotes the nucleotide sequence of SVVP13C insert in construct BaculoFBU/SVVP13C-CO where the SVV P1 region is codon optimized for expression in insect cells (B).

SEQ ID NO:21 denotes the amino acid sequence of SVVP13C polyprotein expressed from BaculoFBU/SVVP13C-CO.

SEQ ID NO:22 denotes BaculoFBU/VVP13C VP3/VP1.

SEQ ID NO:23 denotes the nucleic acid sequence of the SVVP13C VP3/VP1 insert of construct BaculoFBU/SVVP13C VP3/VP1.

SEQ ID NO:24 denotes the nucleic acid sequence of the SVVP13CD insert of construct BaculoFBU/SVVP13CD.

SEQ ID NO:25 denotes the amino acid sequence of SVVP13CD polyprotein expressed from BaculoFBU/SVVP13CD.

SEQ ID NO:26 denotes the nucleic acid sequence of the PCR product containing the coding sequence for SVVP1-His.

SEQ ID NO:27 denotes the amino acid sequence of SVVP1-His expressed from construct BaculoG/SVVP1-His-sIRES-SVV3C.

SEQ ID NO:28 denotes the nucleic acid sequence of the PCR product containing the coding sequence for SVVP1-His that has been codon optimized for expression in insect cells (SVVP1CO-His).

SEQ ID NO:29 denotes the amino acid sequence of SVVP1-His expressed from construct BaculoG/SVVP1CO-His-sIRES-SVV3C.

SEQ ID NO:30 denotes the nucleic acid sequence of the PCR product containing the coding sequence for SVV3C.

SEQ ID NO:31 denotes the amino acid sequence of SVV3C expressed from constructs BaculoG/SVVP1-His-sIRES-SVV3C and BaculoG/SVVP1CO-His-sIRES-SVV3C.

SEQ ID NO:32 denotes the nucleic acid sequence of the SVVP1-His-sIRES-SVV3C expression cassette in pORB-SVVP1-His-sIRES-SVV3C.

SEQ ID NO:33 denotes the nucleic acid sequence of the SVVP1CO-His-sIRES-SVV3C expression cassette in pORB-SVVP1CO-His-sIRES-SVV3C.

SEQ ID NO:34 denotes PCR primer P3219012A (SVVP1 Fwd).

SEQ ID NO:35 denotes PCR primer P3219039A (SVVP1 His Rev).

SEQ ID NO:36 denotes PCR primer P3219012C (SVVP1-CO Fwd).

SEQ ID NO:37 denotes PCR primer P3219039B (SVVP1-CO His Rev).

SEQ ID NO:38 denotes PCR primer P3219012E (SVV3C Fwd).

SEQ ID NO:39 denotes PCR primer P3219039C (SVV3C Rev).

SEQ ID NO:40 denotes PCR primer P3219165A (VP3/VP1 Fwd).

SEQ ID NO:41 denotes PCR primer P3219165B (VP3/VP1 Rev).

SEQ ID NO:42 denotes PCR primer P3219166A (SVV3D Fwd).

SEQ ID NO:43 denotes PCR primer P3219166B (SVV3C Rev).

SEQ ID NO:44 denotes PCR primer P3219166C (SVV3D Rev).

SEQ ID NO:45 denotes SVV3D coding sequence.

The invention further includes the following clauses:

1. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a killed Seneca Valley Virus A (SVA) comprising:

(a) a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or

(b) a nucleic acid sequence 97% identical to SEQ ID NO:1, which encodes a polypeptide having immunologically-effective activity of a polypeptide of SEQ ID NO:3.

2. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a killed SVA comprising:

(a) having the amino acid sequence of SEQ ID NO:3;

(b) having an amino acid sequence 80% identical to SEQ ID NO:3 and having a biological or immunologically-effective activity of a polypeptide encoded by SEQ ID NO:3; or

(c) that is a fragment of the amino acid sequence of SEQ ID NO:3 comprising at least 15 contiguous amino acids of SEQ ID NO:3 and having an immunologically-effective activity.

3. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of a killed SVA comprising:

(a) a nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or

(b) a nucleic acid sequence 97% identical to SEQ ID NO:1 or SEQ ID NO:2, which encodes a polypeptide having immunologically-effective activity of a polypeptide of SEQ ID NO:3.

4. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of an immunogenic compositions according to clause 1.

5. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of an immunogenic compositions according to clause 2.

6. A method of generating an immune response in a mammal, comprising administering an immunologically-effective amount of an immunogenic composition according to clause 3.

7. A method according to clause 1, wherein the mammal is a swine, and the immune response provides protective immunity to disease caused by SVA infection.

8. A method according to clause 2, wherein the mammal is a swine, and the immune response provides protective immunity to disease caused by SVA infection.

9. A method according to clause 3, wherein the mammal is a swine, and the immune response provides protective immunity to disease caused by SVA infection.

10. A vaccine comprising one or more antigens of Senecavirus A (SVA), wherein the SVA is any SVA comprising:

(a) a nucleic acid encoded by SEQ ID NO:1, and/or comprises the sequence of SEQ ID NO:1 and/or comprises the RNA equivalent of SEQ ID NO:1;

(b) a nucleic acid which sequence is at least 99% identical with SEQ ID NO:1 and/or is at least 99% identical with the RNA equivalent of SEQ ID. NO:1;

(c) a polypeptide that is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1 or 2;

(d) a polypeptide that is encoded by a polynucleotide that is at least 80% homologous to or identical with polynucleotides of a);

(e) a protein fragment that is encoded by a polynucleotide that comprises at least 15, preferably 24, more preferably 30, even more preferably 45 contiguous nucleotides included in the sequences of SEQ ID NO: 1 or SEQ ID NO: 2;

(f) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;

(g) a polypeptide that is at least 80% homologous to and/or identical with a polypeptide of f);

(h) a fragment of the polypeptides of f) and/or g);

(i) a polypeptide of f) or g); or

(j) a fragment of h) or i) comprising at least 5, preferably 8, more preferably 10, even more preferably 15 contiguous amino acids included in the sequences of SEQ ID NO: 3.

11. The vaccine of clause 10, wherein the vaccine is recombinant vaccine or a killed vaccine.

12. The vaccine of clause 10, wherein the vaccine is killed vaccine.

13. The vaccine of clause 10, wherein the Senecavirus A (SVA), is chemically inactivated.

14. The vaccine of clause 13, wherein the Senecavirus A (SVA), is chemically inactivated by treatment with a chemical inactivating agent which includes a compound selected from the group consisting of ethylenimine, binary ethylenimine, acetylethylenimine and mixtures thereof.

15. The vaccine of clause 14, wherein the Senecavirus A (SVA), is chemically inactivated by treatment with binary ethylenimine.

16. The vaccine of clause 10, wherein the vaccine further comprises an adjuvant.

17. The vaccine of clause 16, wherein the adjuvant is an EMULSIGEN® oil-in-water emulsion-based adjuvant.

18. The vaccine of clause 6, wherein the Senecavirus A (SVA) comprises SEQ ID NO:1, and/or comprises the RNA equivalent of SEQ ID NO:1.

19. The vaccine of clause 11, wherein the vaccine is recombinant vaccine.

20. The vaccine of clause 11, wherein such recombinant vaccine comprises one or more immunogenic components selected from the group consisting of:

(a) an isolated nucleic acid encoding an antigen of Senecavirus A (SVA), wherein the recombinant polypeptide has at least 90% homology with SEQ ID NO:3,

(b) a vector comprising the isolated nucleic acid of a),

(c) the recombinant protein encoded by the nucleic acid of a), and

(d) any combination thereof.

21. The vaccine of clause 20, wherein such vaccine comprises a pharmaceutical acceptable carrier and/or excipient.

22. The vaccine of clause 21, wherein the excipient is one or more adjuvants.

23. The vaccine of clause 22, wherein the adjuvant is an EMULSIGEN® oil-in-water emulsion-based adjuvant.

24. The vaccine of clause 20, wherein the vaccine further comprises one or more additional antigens.

25. The vaccine of clause 20, wherein an immunogenic component is the isolated nucleic acid.

26. The vaccine of clause 20, wherein an immunogenic component is the vector.

27. The vaccine of clause 11, wherein an immunogenic component is the recombinant Senecavirus A (SVA) P1 protein.

28. The vaccine of clause 20, wherein an immunogenic component is a combination.

EXAMPLES Example 1

This study utilized conventional animals to determine the preliminary feasibility of induction of a serological response following vaccine administration. The primary purpose of this study was to evaluate if inactivated whole-virus preparations using Senecavirus A (SVA) (SEQ ID NO:1 or SEQ ID NO:2, and/or a nucleic acid sequence 97% identical to SEQ ID NO:1, which encodes a polypeptide having immunologically-effective activity of a polypeptide of SEQ ID NO:3) seroconverted in conventional pigs to the SVA vaccine.

For virus isolation, 0.5 mL of vesicular fluid was filtered through a 0.2/0.8 μm syringe filter (Pall Acrodisc Cat 4658) and the filtrate was used for inoculation on to swine testes cells (ST cells). ST cells were grown in 6-well plates to 80-100% confluency. Media was aspirated and 0.25mL of the filtrate was inoculated on to the cells. Following an hour of adsorption at 37° C., plain serum free media was added to cells. Plates were incubated at 37° C., 5% CO2 atmosphere and checked daily for cytopathic effects (CPE). CPE was typically complete in 24-28 hours. Harvested virus was passed several times and was used to generate serials.

The viral stock was prepared by inoculating flasks planted with AI-ST cells with 5mL viral stock and 160 mL of media (Minimum Essential Media+2.5% HEPES). Flasks were incubated for approximately 48 hrs. Flasks were frozen and then thawed at room temperature. Material was 0.2 μm filtered. Viral harvest was inactivated with a combination of 10 mM BEI and 0.2% formaldehyde with constant agitation at 37° C. for 72 hrs. The inactivation was neutralized with sodium thiosulfate (17% of BEI volume added) and sodium bisulfite. Inactivation was confirmed by two passages of material on AI-ST cells. For the initial passage, 10 mL of inactivated material was inoculated onto a T75 flask of AI-ST cells. Flasks were incubated for seven days at 37° C.+5% CO₂ and periodically evaluated for the presence of cytopathic effect. For the second passage, flasks were frozen, then thawed. Material was centrifuged and 10 mL of the supernatant was inoculated onto a T75 flask of AI-ST cells. Flasks were incubated for seven days at 37° C.+5% CO₂ and periodically evaluated for the presence of cytopathic effect. Positive and negative controls samples were included in the assay. Results confirmed lack of growth in both lots. Inactivated viral harvest was concentrated to 12.4× using a 10 k, 650 cm² ultrafiltration hollow fiber cartridge. The concentrated serial was aseptically combined with EMULSIGEN® D (commercially available from Phibro Animal Health Corporation) to achieve a 12.5% formulation. The mixture was stirred for 30 minutes at room temperature and then aseptically dispensed into 30 mL vaccine bottles and stored at 4° C. Material was tested for bacterial sterility by routine culture (anaerobic and aerobic) on blood agar plates at 37° C. for 48 hours. No bacterial contamination was detected.

Twenty pigs were randomized into two groups as shown in Table 1. See Table 1 below for group descriptions and housing structure. On D0, pigs were administered a 2 mL intramuscular dose of the SVA vaccine or a placebo. On D21, animals received a booster administration of the SVA vaccine or placebo. Blood was collected from all pigs prior to administration of the treatment at each vaccination (D0 and D21) and on D35. Subsets of serum samples were assayed for evidence of seroconversion to SVA. General health observations were recorded throughout the study. Injection sites were observed for reactions for a minimum of three days following administration of the vaccine. Animals were humanely euthanized at the end of the trial.

TABLE 1 Study Design Group Room n Vaccine treatment Dose/Route 1 114 11 SVA inactivated prototype 2 m1L/IM vaccine (Senecavirus A; x + 10; 0.2 μm filtered; pre-inactivation titers (two batches were pooled) = 6.95/6.51 log TCID50/m1L; inactivated with 10 mM BEI + 0.2% formaldehyde; neutralized with sodium bisulfite and sodium thiosulfate; 12.4X concentrated (10 kD hollow-fiber); adjuvanted with 12.5% EMULSIGEN ®D) 2 114 7 Placebo (1xPBS (Gibco, 2 m1L/IM catalog#10010-023, 219 2 L#1793111) adjuvanted with 12.5% EMULSIGEN ®D)

This study demonstrated 100% seroconversion (as measured by virus neutralization) to SVA following administration of two doses of the BEI-inactivated SVA prototype adjuvanted with 12.5% EMULSIGEN® D. See Table 2 for the schedule of key events and sample collection.

TABLE 2 Schedule of key events and sample collection Study Day Study Event D-3 Collection of blood from animals D-1 Transfer of animals from study D0 Vaccination #1 Injection site observations for three days following vaccination Collection of blood from animals D14 Collection of blood from animals D21 Vaccination #2 Collection of blood from animals Injection site observations for three days following vaccination* D0-D34 General health observations (lx daily) D34 Necropsy Collection of terminal blood (1 x 250 m1L bottle) from all animals *Note that observations continued until reactions resolved

To avoid bias, treatments were administered on D0 and D21 by personnel not involved with clinical monitoring of the animals On D0, the 2 mL dose of vaccine was administered to healthy pigs into the musculature of the right neck using an appropriately-sized, sterile needle and syringe. On D21, the process was identical with the exception that the injection was given on the left side of the neck. The lot number, dosage amount, animal identification numbers and timing of administration of vaccine material were recorded on the Vaccine Dose Confirmation Record.

During the vaccination period, animals were evaluated daily using a general health observation form. Specifically, if all animals were normal, an N was entered for status. If an abnormal pig was found, an A was entered for status and the specific animal identification number and abnormality was listed. Injection site areas were monitored for the presence of redness, swelling, heat and pain (either present or absent) and size (cm) for a minimum of three days following each vaccination. If lesions were apparent, they were monitored until resolution.

On blood collection dates, three to eight mL of venous whole blood was collected by the Investigator or designee via the anterior vena cava from each pig using an appropriately sized VACUTAINER® needle, a VACUTAINER® needle holder (both commercially available from Becton Dickinson and Company Corporation) and appropriately sized serum separator tubes (SST).

Serum samples were held at 2-8° C. until testing. Processing was completed within 48 hours of receipt. Blood tubes were centrifuged at 1960× g for 10 minutes at 4° C. The serum was separated from the clot by centrifugation and decanted into two screw-cap cryogenic vials labeled with at least study number, day of study, and animal ID. Aliquots were stored at −70° C.±10° C. The samples were stored for a minimum of six months after the completion of this study.

Selected serum samples were tested by a virus neutralizing assay using SVA isolate NAC #20150909.

At the time of off-test, animals were deeply anesthetized and 1×250 mL centrifuge bottle of blood was collected from each animal. The animal was euthanized and the injection site was palpated. All animals were rendered in accordance with the AUP and facility standard operating procedures.

The pig was considered the experimental unit. A list of available pigs born to sow no.'s 9, 116, 787 and 895 (n=30) and from sow no.'s 141 and 145 (n=6) were used for randomization. Specifically, pigs were assigned a random number (using random.org). Pigs were sorted by sow, then random number. Pen assignments were assigned a random number (using random.org). The pen assignments were then sorted by random number and combined with the animal list.

The statistical analyses and data summaries were conducted by the study monitor. All data were imported into JMP® version 11.1.1 for analysis. Data listings and summary statistics by treatment group were generated.

An internally developed virus neutralization assay was used to measure seroconversation in animals following vaccination. No animals had a detectable serological response prior to vaccination. By D35, all vaccinated animals (Group 1) had a detectable response. The assay was run at a 1:80 dilution and animals were considered either positive or negative. See Table 3 below for a summary of the SVA VN results.

TABLE 3 SVA VN results by group and study day Study day Group Description D0 D35 1 SVA inactivated prototype vaccine 0/11 11/11 2 Placebo 0/9 0/9

The primary objective of this study was to evaluate whether conventional pigs seroconvert to SVA following vaccination with SVA vaccine.

In regards to the SVA vaccine, 100% of pigs vaccinated with the inactivated SVA prototype vaccine were able to generate a neutralizing antibody response. In conclusion, this study was able to demonstrate reasonable expectation of efficacy for the prototype inactivated SVA vaccine.

Example 2

Two gene sequences were ordered in the pUCIDT-Amp vector (Integrated DNA Technologies) The SVVP13C gene (SEQ ID NO:18) is native sequence of the full length P1 polyprotein with 2A and partial 2B and 3B sequences connecting the P1 polyprotein and 3C self-cleaving protease. The SVVP13C-CO gene (SEQ ID NO: 20) is an insect codon optimized version of the SVVP13C with the P1, 2A and 2B sequences codon-optimized using the IDT Codon Optimization Tool while the 3B and 3C remained as native sequences. Both genes have a Kozak sequence before the start codon, as well as BamHI and NotI restriction sites at the 5′ and 3′ ends, respectively. The SVVP13C (SEQ ID NO:18) and SVVP13C-CO (SEQ ID NO: 20) inserts were excised from pUCIDT-AMP-SVVP13C and pUCIDT-AMP-SVVP13C-COplasmids by BamHI and NotI digestion, respectively, and ligated into the pVL1393 vector (BD Biosciences). See FIG. 1 for construct design of the native and codon optimized pVL1393 constructs.

PCR amplification was performed to amplify the SVVP1-His (SEQ ID NO:26) and SVVP1CO-His (SEQ ID NO:27) sequence from pIDT-AMP-SVVP13C and pIDT-AMP-SVVP13C-CO respectively using primers that added the coding sequence for a 6× His tag on the 3′ end of each gene (SEQ ID NO: 34, 35, 36 and 37). The SVV3C sequence (SEQ ID NO:30) was amplified from pIDT-AMP-SVVP13C using primers to add a 5′ SpeI site and a 3′ SacI site (SEQ ID: 38 and 39). (See Table 4 for primers).

TABLE 4 Primer Sequences for SVVP1-His-SVV3C  and SVVP1CO-His-SVV3C SEQ ID Primer Sequence (5′ - 3′) NO: P3219012A GGATCCGCCACCATGGGTAATGTTCA 34 (SVVP1 Fwd) P3219039A GCGGCCGCTCAGTGGTGGTGGTGGTG 35 (SVVP1 His  GTGTTGCATCAGCATCTTTTGCTTGT Rev) AGCTGC P3219012C GGATCCGCCACCATGGGCAACG 36 (SVVP1-CO  Fwd) P3219039B  GCGGCCGCTCAGTGGTGGTGGT 37 (SVVP1-CO  GGTGGTGTTGCATAAGCATCTT His Rev) CTGTTTATAGCTACGG P3219012E  ACTAGTATGCAGCCCAACGTGG 38 (SVV3C Fwd) ACATGGGCTTT P3219039C  GAGCTCTCATTGCATTGTAGCC 39 (SVV3C Rev) AGAGGCTCACCGA

TABLE 5 Primer Sequences for SVVP13C  VP3/VP1 and SVVP13CD SEQ ID Primer Sequence (5′ - 3′) NO: P3219165A CTTCCTACGTGCCTCAGGGGG 40 (VP3/VP1 Fwd) TTGACAACGCCGAGACTGGG P3219165B CCCAGTCTCGGCGTTGTCAAC 41 (VP3/VP1 Rev) CCCCTGAGGCACGTAGGAAG P3219166A TACAATGCAAGGACTGATGAC 42 (SVV3D Fwd) TGAGCTAGAGCCTG P3219166B TCAGTCATCAGTCCTTGCATT 43 (SVV3C Rev) GTAGCCAGAG P3219166C GCGGCCGCTCAGTCGAACAAG 44 (SVV3D Rev) GCCCTCCATCT

The SVV3C insert (SEQ ID NO:30) was ligated into the second multiple cloning site (MCS2) of pORB-MCS1-sIRES-MCS2 vector (Allele Biotechnology) using the SpeI and SacI restriction sites. Subsequently, either SVVP1-His or SVVP1CO-His were ligated into MCS1 of pORB-MCS1-sIRES-SVV3C utilizing the BamHI and NotI restriction sites to generate pORB-SVVP1-His-sIRES-SVV3C (SEQ ID NO:32) and pORB-SVVP1CO-His-sIRES-SVV3C (SEQ ID NO:33) respectively.

Primers were designed to mutate the VP3/VP1 cleavage site (nucleotides 1786-1797) of the SVVP13C sequence (SEQ ID NO:18) in pUCIDT-AMP-SVVP13C from FH/ST to PQ/GV (See Table 5 above for primers, SEQ ID NO:40 and 41) using the Lightning Quik Site Directed Mutagenesis kit (Stratagene). The mutated sequence, SVVP13C VP3/VP1 (SEQ ID NO: 22) was excised from pUCIDT-AMP-SVVP13C VP3/VP1 and ligated into the pVL1393 vector to produce pVL1393-SVVP13C VP3/VP1 An additional construct fused the SVV3D coding sequence (SEQ ID NO:45) to the 3′ end of the SVVP13C sequence (SEQ ID NO:18) by overlap extension PCR (OE-PCR) using overlapping primer sets (SEQ ID NO: 34, 42, 43, and 44). The resulting PCR product, SVVP13CD (SEQ ID NO: 24) was cloned into pVL1393 using BamHI and NotI restriction sites. (See FIG. 3 for diagrams of the SVVP13C VP3/VP1 (SEQ ID NO: 22) and SVVP13CD constructs (SEQ ID NO: 24)).

The recombinant SVV capsid constructs in the pVL1393-based plasmids were co-transfected with FlashBAC ULTRA (FBU) baculovirus DNA (Oxford Expression Technologies) into Sf9 cells, whereas the pORB-based plasmids were co-transfected with BaculoGold baculovirus DNA (BD Biosciences) into Sf9 insect cells using the ESCORT transfection reagent (Sigma Aldrich) as per Manufacturer's instructions. Cell culture supernatants from the transfected Sf9 cells were harvested and clarified by centrifugation at 1,000× g for 5 min to pellet the cellular debris. The clarified supernatant was collected, 0.2 μM-filtered and stored as the P1 transfection harvest. Sf9 insect cells were used to generate P2 stocks, and the P2 stocks were then used to generate P3 and P4 amplifications of the SVV constructs for protein expression evaluations in SF+ insect cells. Baculovirus-infected SF+ cells were harvested and clarified at 10,000 × g for 10 min at 4° C. The cultures of baculovirus-infected SF+ cells were sampled daily to monitor total cells/mL, viable cells/mL, percent viability and cell diameter by Vi-Cell analysis. Amplifications were harvested when viability was ≤30% viability or when viable cells were ≤1×10⁶cells/mL. Additional one mL daily samples were collected to evaluate protein expression as the infections progressed and were processed as described above. Collected supernatant and cell pellet samples were stored at −70° C. until evaluation.

Lysis of Insect Cell Pellets to Separate Soluble and Insoluble Fractions

SF+ insect cell culture samples were centrifuged to pellet the cells after which the media was removed and the cell pellets were frozen until lysis. Pellets were re-suspended in lysis buffer containing the following: 20 mM Tris, 1% Triton X-100, Protease Inhibitor Cocktail for His-tagged Proteins (10 μL/mL) and Benzonase (250 units/mL) in de-ionized water with a pH of 7.4. The re-suspended insect cell lysates were vortexed for 10 sec, incubated at room temperature for 5 min, vortexed again for 10 sec than centrifuged at 19,090 × g for 10 min at 4° C. to pellet insoluble material. The soluble lysates were pipetted off the insoluble fractions and stored in tubes at −70° C.

Purification of SVV Recombinant Capsid Proteins

Supernatant harvests containing the expressed recombinant SVV capsid proteins were 0.2 μM filtered, dispensed into ultracentrifuge tubes, and centrifuged at 100,000 × g for two hours at 4° C. to pellet protein and possible VLPs. The clarified supernatant was carefully decanted and the pelleted material was re-suspended in TBS and stored at 4° C. Discontinuous 10%-60% sucrose gradients were used to further purify recombinant SVV capsid proteins for the respective constructs. The respective re-suspended materials were added to the top of the gradient and centrifuged at 100,000 × g for two hours at 4° C. Fractions from the sucrose gradients were collected equally into tubes (with fraction 1 starting at the top of the gradient surface) and stored at 4° C.

SDS-PAGE & Western

SDS-PAGE was performed using the NuPAGE electrophoresis system and 4-12% Bis-Tris MES mini gels. Samples were separated under reducing conditions using 0.05M DTT at 175V for the appropriate time. Gels were stained for total protein using an eStain 2.0 Protein Staining Device or transferred to nitrocellulose membranes using the iBlot system for Western blots. Western blots were performed with anti-SVV peptide rabbit polyclonal antibodies (anti-VP1-2, anti-VP2-2, and anti-VP3-1, varying dilutions) and goat anti-rabbit peroxidase-labeled secondary antibody (1:500) by the Snap ID Protein Detection System (EMD Millipore), utilizing negative control baculovirus antigen in the antibody diluent, and developed using TMB membrane peroxidase substrate.

Dialysis and Concentration of Purified SVV Recombinant Capsid Proteins

Sucrose gradient fractions containing the recombinant protein as determined by Western blot were pooled together and dispensed into a 10,000 MWCO or 50,000 MWCO cellulose membrane dialysis cassette. The dialysis cassette was placed in 3.5 L of TBS with a magnetic stir bar, covered, and placed on a stir plate at 4° C. for a minimum of 6 hrs. The dialysis cassette was then placed into a fresh beaker of 3.5 L of TBS, and further dialyzed with stirring overnight or longer. The sample was removed from the dialysis cassette and concentrated, if needed, depending on the volume of the dialyzed sample. Concentration was performed using a size-exclusion filter unit and centrifugation according to Manufacturer's directions until desired sample volume was achieved.

Electron Microscope (EM) Imaging

The sucrose gradient-purified and dialyzed recombinant SVV capsid protein samples were evaluated by transmission electron microscopy (EM) at the USDA NADC Facility for the visualization of VLPs. The expected size of the icosahedral viral capsid for native SVV is approximately 27 nm in diameter.

Results and Discussion

The native SVV P1 polyprotein (SEQ ID NO:19, amino acids 1-859) is processed by a self-cleaving protease to form the individual viral capsid subunit proteins, VP1-VP4. The SVV baculovirus constructs described in this invention were designed in a manner similar to that used for FMDV baculovirus constructs utilizing self-cleavage of the expressed polyprotein into individual protein subunits for formation of an empty capsid or VLP. Constructs encoding full length SVV P1 polyprotein, 2A, partial 2B and 3B, and 3C (SEQ ID NO:18) were prepared with native coding sequence or with the P1, 2A and 2B sequences codon-optimized (SEQ ID NO:20) for insect cells. Previous research with the similar pVL1393-based FMDV baculovirus constructs revealed SF+ cell toxicity attributed to 3C expression. Concern as to potential SVV 3C toxicity was addressed by also generating pORB-based baculovirus constructs for evaluation. The pORB-based constructs utilize an internal ribosomal entry site (WSSV sIRES) (SEQ ID NO:32, nucleic acids 2647-2826) allowing for the initiation of cap-independent mRNA translation, which differs from the 5′ cap dependent translation of the sequence most adjacent to the 3′-end of the polyhedrin promoter. Several studies have shown that the use of a sIRES, such as that from WSSV, has often resulted in decreased protein expression of the translated sequence placed after the sIRES. In the context of the described SVV baculovirus constructs (SEQ ID NOs:32 and 33), the utilization of the sIRES may reduce expression of the SVV 3C self-cleaving protease, which in turn may alleviate 3C toxicity issues without reducing expression of SVVP1. The pORB-based and pVL1393-based constructs were similar in that they both utilized the same SVVP1 sequence and included a C-terminal 6×-His tag. However, the key difference between the pORB- or pVL1393-based constructs was that the pORB-based constructs placed the SVV3C protease sequence behind the sIRES site. After evaluations of these initial sets of constructs, two more constructs were created modifying the original SVV DNA sequence. One construct modified the VP3/VP1 cleavage site sequence (SEQ ID NO:22) and the other construct further incorporated the SVV3D DNA sequence at the end of the SVV3C sequence (SEQ ID NO:24). All constructs were evaluated for expression of the capsid subunits as well as for the production of VLPs.

Expression of SVV Capsid Proteins in Baculovirus—Infected Insect Cells

The BaculoFBU/SVVP13C (SEQ ID NO:18) and BaculoFBU/SVVP13C-CO (SEQ ID NO:20) constructs were used to infect SF+ cells with samples collected to evaluate the expression of SVV capsid proteins. Protein bands of expected sizes for VP1 (SEQ ID NO:19, amino acids 596-859), VP2 (SEQ ID NO:19, amino acids 73-356) and VP3 (SEQ ID NO:19, amino acids 357-595) capsid subunits were detected by Western blot with anti-SVV P1 subunit-specific antibodies in the supernatant from the baculovirus-infected insect cells, which were similar in size to native SVV capsid proteins (FIG. 4). Interestingly, an additional protein band of ˜55 kDa was detected in the alpha-SVV VP1 and alpha-SVV VP3 Western blots. The ˜55 kDa band was not detected in the native SVV antigen sample or the negative control (FIG. 4). The presence of the additional protein of ˜55 kDa in size suggests that it may comprise an uncleaved VP3-VP1 protein product.

FIG. 4 also provides a comparison of SVV capsid protein expression levels between the native (A) (SEQ ID NO:18) and the codon-optimized (B) (SEQ ID NO:20) SVV DNA sequences in baculovirus-infected SF+ cells. Based on these Western blots, there was no apparent difference in SVV capsid protein expression levels between the codon-optimized BaculoFBU/SVVP13C-CO (SEQ ID NO:20) and the original, native BaculoFBU/SVVP13C (SEQ ID NO:18), therefore only the BaculoFBU/SVVP13C (SEQ ID NO:18) construct was evaluated further. The second set of constructs with the 3C protease placed behind the sIRES did not produce detectable SVV capsid subunit proteins during infection of SF+ insect cells. Not only did the sIRES constructs not produce detectable levels of the SVV capsid subunit proteins, no SF+ cytotoxicity issues were observed. Consequently, the sIRES constructs were not further evaluated. Based on these overall observations, the supernatant harvest for BaculoFBU/SVVP13C-infected SF+ insect cells was the most promising construct to move forward for sucrose gradient purification and evaluation for the presence of VLPs.

To determine the expected mobility of BaculoFBU/SVVP13C-derived SVV capsid proteins in a sucrose gradient, pelleted native SVV virus was separated on a sucrose gradient and analyzed by Western blot (FIG. 5A). The majority of native SVV subunit proteins were detected in fractions 5 and 6 after sucrose gradient purification for native SVV. It was anticipated that if VLPs were formed in the baculovirus-infected SF+ cells they would present in a similar or slightly higher range of collected gradient fractions.

Harvest supernatant from the BaculoFBU/SVVP13C construct was also processed for sucrose gradient purification. Western blots of the sucrose gradient fractions only detected a small portion of the recombinant SVV VP2 capsid protein in sucrose fraction one (FIG. 5B). VP1 and VP3 were not detected in the sucrose fractions, although a faint protein band at ˜55 kDa, thought to be uncleaved VP3-VP1, was detected throughout the sucrose fractions by α-SVV VP1 Western blot. No recombinant SVV capsid proteins were detected in fractions 5 and 6 in contrast to the detection of expected proteins from native SVV. These results suggest that the capsid subunit proteins expressed in BaculoFBU/SVVP13C-infected insect cells do not form VLPs.

Even though BaculoFBU/SVVP13C (SEQ ID NO:18) produced recombinant SVV capsid proteins, detected as subunit proteins with alpha-SVV antibodies, VLPs were not observed. The capsid subunit proteins were of similar size when compared to the native SVV virus proteins, but several other protein bands were also detected in the baculovirus-infected SF+ cells, including a protein band at ˜55 kDa in the alpha-SVV VPI and alpha-SVV VP3 Western blots, suggesting an uncleaved VP3-VP1 protein product not present in the native SVV virus. This may be an indication of an issue with the efficient cleavage and separation of the VP3-VP1 subunits which in turn may hinder VLP formation in baculovirus-infected insect cells.

Evaluation of Modified BaculoFBU/SVVP13C Constructs

To investigate the possibility of the SVV capsid subunit proteins VP1 and VP3 not separating completely, two new constructs were designed based on previous publications. The 2008 publication by Hales et al. stated that the SVV P1 VP3/VP1 cleavage site, FH/ST, was atypical of picornaviruses including the genus most closely related to SVV, Cardiovirus. In comparison, the typical cleavage site of PQ/GV is conserved in many known picornaviruses. Therefore, mutation of the VP3/VP1 cleavage site to a typical picornavirus cleavage sequence may enhance cleavage of the VP3 and VP1 capsid subunits. The BaculoFBU/SVVP13C (SEQ ID NO:18) construct was mutated to contain a PQ/GV sequence at the VP3/VP1 interface resulting in a construct designated as BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22). This new construct was used to infect SF+ cells and evaluated in protein expression assessments in a similar fashion as to that of the BaculoFBU/SVVP13C construct (SEQ ID NO:18). Western blot analysis of BaculoFBU/SVVP13C VP3/VP1-derived supernatant samples using alpha-SVV VP1 or alpha-SVV VP3 antibodies detected the putative uncleaved VP3-VP1 protein product and individual subunits of VP3 or VP1 bands in the same proportion as was observed from BaculoFBU/SVVP13C (SEQ ID NO:18) (FIG. 6). A protein band at the expected full length of the SVV P1 polyprotein, ˜95 kDa, was also detected in the BaculoFBU/SVVP13C VP3/VP1 harvest supernatants.

Additionally, Western blot evaluations of the sucrose gradient fractions of the harvest supernatant (FIG. 7) were comparable to the results of BaculoFBU/SVVP13C (SEQ ID NO:18). The α-SVV VP2 Western blot detected the recombinant SVV VP2 capsid protein throughout the sucrose fractions with the majority in fractions one and nine. Subunit VP1 and VP3 proteins were not detected in the sucrose fractions, although a protein band at ˜55 kDa was detected throughout the sucrose fractions of the α-VP1 Western and in sucrose fractions one and nine of the α-VP3 Western that is possibly the VP3-VP1 uncleaved proteins. All three Westerns had several protein bands detected in the very last sucrose fraction that appeared similar to the starting sample of the sucrose gradient. This suggests the SVV capsid proteins may aggregate and pellet to the bottom of the gradient. Capsid proteins were not detected in the last fraction of the BaculoFBU/SVVP13C (SEQ ID NO:18) sucrose gradient Westerns in FIG. 5b , but were observed in previous evaluations (NB 3219-123). It appears from these Western evaluations that the mutation of the cleavage sequence from FH/ST to PQ/GV had no effect on the presence of the ˜55 kDa band thought to be VP3-VP1 uncleaved. Compared to the original construct no increase in the amount of subunit VP3 and VP1 protein was detected and no VLP formation was observed.

Other genera within picornavirus including enteroviruses and apthoviruses have shown more efficient cleavage of VP3-VP1 when having the 3CD protease in the native virus; therefore, a second Senecavirus construct, BaculoFBU/SVVP13CD (SEQ ID NO:24), was designed. BaculoFBU/SVVP13CD (SEQ ID NO:24) was expressed in SF+ cells and the supernatant harvest was evaluated by Western blot (FIG. 8). Results comparable to the previous VLP assessments were observed, with detection of VP1, VP2 and VP3 subunits as well as the suspected uncleaved VP3-VP1 protein of ˜55 kDa in the α-SVV VP1 and α-SVV VP3 Western blots.

In contrast to SVV protein expression assessments with other SVV baculovirus constructs, Western blot evaluations of the sucrose fractions for BaculoFBU/SVVP13CD (SEQ ID NO:24) exhibited monomeric SVV VP1, VP2 and VP3 capsid subunit proteins present in sucrose fractions 1 and 2 with VP1 and VP2 detected throughout the sucrose fractions. The ˜55 kDa protein band was present in the α-SVV VP1 and α-SVV VP3 Western blots in sucrose fractions 1 and 2, but not in the negative control sample or α-SVV VP2 Western blots (FIG. 9). Similar to the BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) sucrose fraction evaluations, a majority of the SVV capsid proteins aggregated and pelleted to the bottom of the sucrose gradient for BaculoFBU/SVVP13CD (SEQ ID NO:24). However, detection of SVV VP1 and VP2 subunit proteins throughout the fractions derived from BaculoFBU/SVVP13CD-infected SF+ cells construct suggest the possibility of VLP formation.

Despite unclear results from the sucrose gradient evaluations of each construct, fractions expected to contain VLPs were evaluated by electron microscopy (EM) at the USDA NADC. Pooled sucrose fractions from supernatant harvests BaculoFBU/SVVP13C (SEQ ID NO:18), BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) and BaculoFBU/SVVP13CD (SEQ ID NO:24) were dialyzed in TBS and concentrated in preparation for EM imaging. The samples had high background making it difficult to clearly depict VLPs by EM negative staining. Some spherical shapes were seen sparsely in the BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) and BaculoFBU/SVVP13CD (SEQ ID NO:24) pooled sucrose fractions that were similar in size to expected SVV VLPs, but there were not enough of them throughout the sample to confirm the presence of VLPs.

Recombinant SVV Proteins are Expressed Inside the SF+ Insect Cells

One possibility as to why the individual recombinant SVV viral capsid proteins are detected in the supernatant, but do not appear to form VLPs is that VLPs might disassociate shortly after release into the supernatant due to the low pH of the insect cell media. To test this hypothesis, cell pellet samples from day 3, when viability of the cells was still relatively high, of BaculoFBU/SVVP13C (SEQ ID NO:18), BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) and BaculoFBU/SVVP13CD (SEQ ID NO:24) infections were lysed in physiological pH buffer to obtain the soluble protein fraction for evaluation of recombinant SVV subunit proteins by Western blot (FIG. 10). As seen with the supernatant harvest samples of the baculo SVV constructs, there were detectable levels of subunit capsid proteins VP1, VP2 and VP3 in the Day 3 soluble fractions. An ˜55 kDa band suspected to be uncleaved VP3-VP1 proteins was also detected in the α-SVV VP1 and α-SVV VP3 Westerns in the baculo SVV sample lanes as observed previously. The Day 3 soluble fraction samples were sucrose gradient purified and evaluated by Western blot to observe if VLPs were present in the cells before lysis (FIG. 11).

The sucrose fractions from the Day 3 soluble fractions had comparable results as seen with the supernatant harvest sucrose fractions from insect cells infected with BaculoFBU/SVVP13C (SEQ ID NO:18), BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) and BaculoFBU/SVVP13CD (SEQ ID NO:24), respectively. The protein bands expected to be the SVV capsid proteins were detected mostly in fractions one and two and/or pelleted to the bottom suggesting the SVV subunit proteins inside the insect cells do not form VLPs prior to cell lysis. EM was performed on pooled sucrose fractions of BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) Day 3 soluble sample with no VLPs being observed for this sample. BaculoFBU/SVVP13C (SEQ ID NO:18) and BaculoFBU/SVVP13CD (SEQ ID NO:24) Day 3 soluble sucrose fractions were not evaluated by EM.

CONCLUSIONS

Although it was determined that recombinant SVV viral capsid proteins VP1, VP2 and VP3 were expressed to some extent as fully cleaved proteins, they did not result in VLP formation based on sucrose gradient purification and EM imaging. In the Western blot evaluations of sucrose gradient fractions, the recombinant SVV capsid proteins were detected in the first few fractions and/or in the last fractions of the gradient. These results suggest that the proteins do not assemble into VLPs, but rather remain as non-associated monomers or form large aggregates indicative of misfolded or misassembled proteins. Electron microscopy supports these results as VLPs were not detected in the pooled sucrose fractions which contained the SVV capsid subunit proteins.

Example 3

This study utilizes conventional animals to determine the preliminary feasibility of induction of a serological response following vaccine administration. The primary purpose of this study is to evaluate whether administration of prototype vaccines BaculoFBU/SVVP13C (SEQ ID NO:18), BaculoFBU/SVVP13C VP3/VP1 (SEQ ID NO:22) and BaculoFBU/SVVP13CD (SEQ ID NO:24) results in seroconversion in conventional pigs.

Forty pigs are randomized into four groups as shown in Table 1. See Table 6 below for group descriptions and housing structure. On D0, pigs are administered a 2 mL intramuscular dose of the prototype vaccines or a placebo. On D21, animals receive a booster administration of the prototype vaccines or placebo. Blood is collected from all pigs prior to administration of the treatment at each vaccination (D0 and D21) and on D35. Subsets of serum samples are assayed for evidence of seroconversion to SVA. General health observations are recorded throughout the study. Injection sites are observed for reactions for a minimum of three days following administration of the vaccine. Animals are humanely euthanized at the end of the trial. See Table 7 for the schedule of key events and sample collection.

TABLE 6 Study Design Group n Vaccine treatment Dose/Route 1 10 Bacu1oFBU/SVVP13C (SEQ ID NO:18); 2 mL/IM inactivated with 5-10mM BEI; adjuvanted with 12.5% EMULSIGENOD) 2 10 Bacu1oFBU/SVVP13C VP3/VP1 2 mL/IM (SEQ ID NO:22) inactivated with 5-10mM BEI; adjuvanted with 12.5% EMULSIGENOD) 3 10 BaculoFBU/SVVP13CD (SEQ ID NO:24); 2 mL/IM inactivated with 5-10mM BEI; adjuvanted with 12.5% EMULSIGENOD) 4 10 Placebo (BaculoFBU/empty); inactivated 2 mL/IM with 5-10 mM BEI; adjuvanted with 12.5% EMULSIGENOD)

TABLE 7 Schedule of key events and sample collection Study Day Study Event D-3 Collection of blood from animals D-1 Transfer of animals from study D0 Vaccination #1 Injection site observations for three days following vaccination Collection of blood from animals D14 Collection of blood from animals D21 Vaccination #2 Collection of blood from animals Injection site observations for three days following vaccination* D0-D34 General health observations (lx daily) D34 Necropsy Collection of terminal blood (1 x 250 m1L bottle) from all animals *Note that observations continued until reactions resolved

To avoid bias, treatments are administered on D0 and D21 by personnel not involved with clinical monitoring of the animals. On D0, the 2 mL dose of vaccine is administered to healthy pigs into the musculature of the right neck using an appropriately-sized, sterile needle and syringe. On D21, the process is identical with the exception that the injection was given on the left side of the neck. The lot number, dosage amount, animal identification numbers and timing of administration of vaccine material are recorded on the Vaccine Dose Confirmation Record.

During the vaccination period, animals are evaluated daily using a general health observation form. Specifically, if all animals are normal, an N is entered for status. If an abnormal pig is found, an A is entered for status and the specific animal identification number and abnormality is listed. Injection site areas are monitored for the presence of redness, swelling, heat and pain (either present or absent) and size (cm) for a minimum of three days following each vaccination. If lesions are apparent, they are monitored until resolution.

On blood collection dates, three to eight mL of venous whole blood are collected by the Investigator or designee via the anterior vena cava from each pig using an appropriately sized VACUTAINER® needle, a VACUTAINER® needle holder (both commercially available from Becton Dickinson and Company Corporation) and appropriately sized serum separator tubes (SST).

Serum samples are held at 2-8° C. until testing. Processing is completed within 48 hours of receipt. Blood tubes are centrifuged at 1960 × g for 10 minutes at 4° C. The serum is separated from the clot by centrifugation and decanted into two screw-cap cryogenic vials labeled with at least study number, day of study, and animal ID. Aliquots are stored at −70° C.±10° C. The samples are stored for a minimum of six months after the completion of this study.

Example 4

This study utilized conventional animals to evaluate the efficacy of a two-dose, Senecavirus A vaccine, inactivated, whole virus against a heterologous challenge with a heterologous Senecavirus A field isolate. A total of 25 pigs were used for the study. Animals were randomized into two treatment groups. On D0, thirteen pigs in the SVA-Vx group were inoculated intramuscularly (IM) with Senecavirus A vaccine, inactivated, whole virus, while the remaining twelve pigs in the Placebo group received a control product. On D14, a booster vaccine was administered intramuscularly to all pigs using the appropriate material. On D35, all pigs were challenged with a total volume of 5 mL (2 mL orally and 3 mL intranasally) of 8.36 log TCID50/dose heterologous Senecavirus A field isolate (viral harvest). All pigs were housed comingled within one room. Pigs were monitored daily for general health from D0 through D33. From D34 through D49, pigs were monitored daily for clinical signs associated with SVA infection. Blood and rectal temperatures were taken periodically throughout the study. All animals were necropsied on D49 (14 days post challenge). See Table 8 for experimental design summary.

TABLE 8 Study design Vaccine Off- Group n Room (D0, D14) Challenge (D35) test SVA-Vx 13 316 Senecavirus A 8.36 log D49 vaccine, TCID50/5mL inactivated, dose; whole virus heterologous Placebo 12 316 Placebo Senecavirus A field isolate (viral harvest)

See Table 9 below for a summary of the vaccine and control product formulations. Routine culture and a Mycoplasma sp. PCR were performed on the vaccine material; no growth (anaerobic or aerobic on blood agar) or Mycoplasma DNA contamination was detected. The vaccines were administered on D0, intramuscularly into the right side of the neck, midway between the base of the ear and point of the shoulder, using appropriately-sized sterile needles and syringes. On D14, the vaccine was administered in the same location as previously described but on the left side of the neck. All groups received a 2 mL dose.

TABLE 9 Vaccine and Control Group Treatment SVA-Vx Senecavirus A; pre-MSV; 0.2um filtered; pre-inactivation titer = 7.71 log TCID50/mL; inactivated with 10mM BEI +0.2% formaldehyde; neutralized with sodium bisulfite and sodium thiosulfate; adjuvanted with 12.5% Emulsigen D; Lot+1903423-022. Placebo Mock infected media; inactivated with 10mM BEI +0.2% formaldehyde; neutralized with sodium bisulfite and sodium thiosulfate; adjuvanted with 12.5% Emulsigen D; Lot#3423-023.

Based on reports from field cases and previous publications, animals were monitored for lameness, hoof lesions, and the presence of vesicles. If an animal had any clinical abnormality throughout the study, it was considered affected. Table 10 reports the frequency of affected animals by group. The preventative fraction estimate 0.322 (0.004, 0.539; 95% CI) indicates vaccination reduced the number of affected animals. Mitigated fraction analysis was also done on the number of days an animal displayed an abnormal clinical sign. The mitigated fraction estimate of 0.710 (0.333, 0.935; 95% CI) indicates vaccination reduced the number of days an animal was considered affected.

TABLE 10 Frequency distribution of presence/absence of clinical signs by group during the challenge phase Affected?* No Yes Group N % N % total n SVA-Vx 4 31 9 69 13 Placebo 0 0 11 100 11

*Affected = a clinical sign was observed at least once during the study

Animal? #648 was removed from the analysis

Rectal temperatures were collected during the challenge period; least square mean rectal temperatures by study day and group are presented in FIG. 12. Vaccination resulted in significantly lower temperatures at D40 and 42.

Serum samples from D0, 14, 35, and 49 were evaluated by a virus neutralization assay. Geometric mean titers by group and study day are presented in Table 11. Following two doses of vaccine, 13/13 (100%) of animals had neutralizing titers greater than 400.

TABLE 11 Summary statistics for virus neutralization titers by study day and group. Values of >40 were reported as 20; values <2560 were set to 2560. Study Geometric % animals Group Day n Mean Min Max positive (>40) SVA-Vx 0 13 20.00 20  20 0.00 14 13 68.17 20  160 69.23 35 13 440.64 80 1280 100.00 49 13 640.76 80 2560 100.00 Placebo 0 12 20.00 20  20 0.00 14 12 20.00 20  20 0.00 35 12 21.19 20  40 0.00 49 11 2256.9 640  2560 100.00

The presence of viral RNA in the serum was detected by a qRT-PCR assay. FIG. 13 displays group median quantities (log₁₀ genomic copies/mL) of SVA RNA in serum. SVA RNA was not detected in vaccinated animals at any time during the study. In comparison, viremia was detected in the placebo group from D36 through D42.

In conclusion, vaccination with two doses of Senecavirus A vaccine, inactivated, whole virus, resulted in complete reduction of viremia, a statistically significant reduction in clinical signs (PF=0.322; 95% CI=0.004, 0.539), and greater than four-fold virus neutralization titers in 13/13 vaccinated animals prior to challenge.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the following claims. 

1-8. (canceled)
 9. An antibody that specifically binds to a polypeptide of a recombinant or chemically inactivated virus comprising an amino acid sequence that: (i) is encoded by a polynucleotide comprising 99% sequence identity to SEQ ID NO: 1 and/or 95% sequence identity to SEQ ID NO:2; (ii) has an amino acid sequence of SEQ ID NO: 3; (iii) corresponds to a P1-2A-P3 polypeptide and is encoded bv a polynucleotide comprising 90% sequence identity to SEQ ID NO: 18, 20, 22, 24, 32, and/or 33; and/or (iv) has at least 90% sequence identity to SEQ ID NO: 19, 21, 23, 25, 27, and/or
 29. 10-14. (canceled)
 15. A method for detecting Senecavirus A (SVA) in a sample, comprising: (i) contacting the sample with the antibody of claim 9, and detecting for binding of the SVA to the antibody
 16. A method for diagnosing a Senecavirus A (SVA) infection in a swine, comprising contacting a sample from the swine with a probe specific for a nucleic acid of a recombinant or chemically inactivated virus comprising a polynucleotide, a complement of the polynucleotide, or a DNA equivalent of the polynucleotide or the polynucleotide complement, wherein the polynucleotide: (i) comprises 99% sequence identity to SEQ ID NO: 1; (ii) comprises 95% sequence identity to SEQ ID NO: 2: and/or (iii) encodes a polypeptide having an amino acid sequence of SEQ ID NO: 3; detecting for binding of SVA to the probe: and determining, based on the detection step, whether the swine is infected with SVA
 17. An immunogenic composition comprising the antibody of claim 9 and a pharmaceutically acceptable carrier and/or excipient.
 18. A vaccine comprising the antibody of claim 9 and a pharmaceutically acceptable carrier and/or excipient.
 19. The immunogenic composition of claim 17 wherein the virus is Senecavirus A (SVA).
 20. The immunogenic composition of claim 19 wherein the SVA is chemically inactivated.
 21. The vaccine of claim 18 wherein the virus is Senecavirus A (SVA).
 22. The vaccine of claim 21 wherein the SVA is chemically inactivated. 